Benzofuran derivatives

ABSTRACT

Certain benzofuran derivatives are useful in the treatment of certain ischemic or inflammatory conditions, as well as neuroinflammation, neurodegeneration or degenerative diseases in which mitochondrial dysfunction leads to tissue degeneration. They are also useful in the manufacture of pharmaceutical formulations for the treatment of such conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of non-provisionalapplication Ser. No.10/361,141 filed Feb. 6, 2003 claiming priorityunder 35 U.S.C. 119(e) to U.S. Provisional Applications Serial No.60/355,331 filed on Feb. 7, 2002, and U.S. Provisional ApplicationSerial No. 60/429,584 filed on Nov. 27, 2002, incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The present invention is directed to methods for treatingneuroinflammation, neurodegeneration, or degenerative diseases in whichmitochondrial dysfunction leads to tissue degeneration.

[0003] The invention relates to certain novel compounds havingcytoprotective and neuroprotective activity, and particularly to aseries of benzofuran derivatives. The invention is directed toformulations and methods for treating stroke, myocardial infarction andchronic heart failure, as well as other oxidative stress-relatedconditions that are typically responsive to cellular enzyme modulation.The invention is also directed to a method of treating inflammation byreducing C-reactive protein (CRP).

BACKGROUND INFORMATION

[0004] The invention is concerned with cytoprotective andneuroprotective compounds, which are benzofuran derivatives, saidderivatives including steroisomers, mixtures of stereoisomers andtherapeutically acceptable salts thereof.

[0005] The present invention is concerned with agents providingprotection against mitochondrial dysfunction, neuroinflammation, orneurodegeneration. Compositions of the invention are active in certainexperimental models that predict efficacy in degenerative diseases inwhich mitochondrial dysfunction leads to tissue degeneration eitherdirectly or indirectly, and in diseases involving neuroinflammation andneurodegeneration. Some of the diseases include, but are not limited toAlzheimer's disease, drug-induced Parkinsonism or Parkinson's disease,Friedreich's ataxia (FRDA) and other ataxias, Leber's hereditary opticneuropathy, epilepsy, and myodegenerative disorders such as MELAS(mitochondrial encephalophaty, lactic acidosis and stroke) and MERRF(myoclonic epilepsy ragged red fiber syndrome).

[0006] Compositions of the invention are active in certain experimentalmodels that predict efficacy in, for example, certain ischemic orinflammatory conditions, including but not limited to stroke, myocardialinfarction, congestive heart failure, and skin disorders characterizedby inflammation or oxidative damage. The invention is therefore relatedto the use of the cytoprotective derivatives in such conditions.

[0007] 2,3-Dihydro-5-oxy-4,6,7-trimethyl-2-optionally substituted alkylbenzofurans have been disclosed as antioxidizing pharmaceutical productshaving anti-ischemic properties in U.S. Pat. No. 5,114,966. Hydroxaminederivatives of 2,3-dihydrobenzofuran carboxy acids have been disclosedin U.S. Pat. No. 5,480,645. 2,3-Dihydrofuran derivatives useful inpreventing and treating neovascularization have been disclosed in U.S.Pat. No. 5,719,167 and U.S. Pat. No. 5,798,356. 5-Hydroxybenzofuranshave been disclosed for the treatment of a pathological cellproliferative disease in U.S. Pat. No. 5,674,876. A method of inhibitingmammalian leukotriene biosynthesis with 6-hydroxybenzofurans has beendisclosed in U.S. Pat. No. 4,714,711.

[0008] While various agents have heretofore been provided for suchconditions, it has, however, remained desired to provide new therapiesfor conditions characterized by oxidative stress, and particularly, forproviding protection in the event of cerebral ischemia and inflammation;especially desired are agents that are effective even if firstadministered after a significant period of time (e.g., about 5 or morehours) following an ischemic or oxidative insult.

[0009] Furthermore, there is a need for compounds, compositions, andmethods that limit or prevent damage to organelles, cells and tissuesinitiated by various consequences of mitochondrial dysfunction,neuroinflammation or neurodegeneration. Such agents would be suitablefor the treatment of degenerative diseases particularly mitochondriaassociated diseases. The present invention fulfills these needs.

SUMMARY OF THE INVENTION

[0010] One aspect of the present invention is concerned with certaincompounds that are effective in the treatment of diseases associatedwith mitochondrial dysfunction, as well as neuroinflammatory orneurodegenerative diseases, including but not limited to Alzheimer'sdisease, diabetes mellitus, drug-induced Parkinsonism or Parkinson'sdisease, neuronal and cardiac ischemia, Huntington's disease,Friedreich's ataxia and other cerebellar ataxias, Leber's hereditaryoptic neuropathy, schizophrenia, MELAS, MERRF, and other myodegenerativedisorders, comprising administering to a subject in need of treatment aneffective amount of a compound having the structure of Formula I orFormula II, as described herein.

[0011] It has surprisingly been found that certain compounds of thisinvention limit or prevent damage to organelles, cells and tissuescaused by mitochondrial dysfunction, oxidative stress, orneuroinflammation as demonstrated by providing protection in standardexperimental models of mitochondria dysfunction caused by MPP⁺(1-methyl-4-phenylpyridinium) or oxidative stress caused by beta-amyloidor high glutamate.

[0012] Certain compounds are particularly active in restoring orpreserving metabolic integrity in oxidatively competent cells that havebeen subjected to oxygen deprivation. Such compounds, predominantlybenzofuran derivatives are useful in the manufacture of pharmaceuticalcompositions for treating a number of conditions characterized byoxidative stress, and particularly, in providing protection in the eventof cerebral ischemia, ultraviolet exposure, or inflammation, even whenadministered a significant time interval after an ischemic or oxidativeinsult. In particular, the compositions of the present invention areuseful in the treatment of stroke, as demonstrated by providingneuroprotection in a standard experimental model of focal cerebralischemia. They are also useful in the treatment of myocardial ischemia(myocardial infarction), as well as other indications characterized byoxidative stress and/or inflammation, including, but not limited to,diabetes, renal disease, pre-menstrual syndrome, asthma, cardiopulmonaryinflammatory disorders, chronic heart failure, rheumatoid arthritis,muscle fatigue, intermittent claudication, and for the preservation ofallograft tissue for transplantation. Particularly with regard todermatological conditions, the compounds, formulations and methods ofthe present invention are useful in regulating skin condition,regulating the signs of skin aging, and in treating a number ofconditions, including, but not limited to preventing and protecting skintissue against age-related damage or damage resulting from insults suchas harmful (UV) radiation, stress and fatigue.

[0013] The present invention concerns the compounds represented by theFormula I:

[0014] wherein:

[0015] R¹ is: hydrogen, optionally substituted (C₁-C₆)-alkyl, optionallysubstituted (C₂-C₁₀)-alkenyl, optionally substituted aryl, (optionallysubstituted (C₁-C₆)-alkoxy)carbonyl, or halogen;

[0016] R² and R³ are independently selected from optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₂-C₁₀)-alkenyl, or optionallysubstituted (C₃-C₈)-cycloalkyl;

[0017] R⁴ is: hydrogen, optionally substituted aryl, (optionallysubstituted(C₁-C₆)-alkyl)carbonyl, (optionally substitutedaryl)carbonyl, (optionally substituted heterocyclyl)carbonyl,(optionally substituted heterocyclylalkyl)carbonyl, (optionallysubstituted (C₁-C₆)-alkoxy)carbonyl, (optionally substituted(C₂-C₁₀)-alkenyloxy)carbonyl, (optionally substituted amino)carbonyl,carboxy, formyl, or hydroxy(optionally substituted)(C₁-C₆)-alkyl;

[0018] R⁵ is: hydrogen, (C₁-C₆)-alkyl, (C₂-C₁₀)-alkenyl, (optionallysubstituted alkoxy)carbonyl, carboxy, (optionally substitutedamino)carbonyl, or optionally substituted aryl; provided that one of R⁴or R⁵ is hydrogen, and that when R⁴ is hydrogen R⁵ is not hydrogen, andwhen R⁵ is hydrogen R⁴ is not hydrogen; and

[0019] R is: hydrogen, (C₁-C₆)-alkyl, (C₁-C₆)-alkylcarbonyl, phosphoryl,or polyalkoxy; or

[0020] R and R¹ with the atoms to which they are attached form anoptionally substituted ring; and single stereoisomers, mixtures ofstereoisomers, and the pharmaceutically acceptable salts thereof.

[0021] The present invention also concerns the compounds represented bythe Formula II:

[0022] wherein:

[0023] R⁶ is: hydrogen, optionally substituted (C₁-C₆)-alkyl, optionallysubstituted (C₂-C₁₀)-alkenyl, optionally substituted aryl, (optionallysubstituted (C₁-C₆)-alkoxy)carbonyl, or halogen;

[0024] R⁷ and R⁸ are independently selected from optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₂-C₁₀)-alkenyl, or optionallysubstituted (C₃-C₈)-cycloalkyl;

[0025] R⁹ is: optionally substituted aryl, (optionallysubstituted(C₁-C₆)-alkyl)carbonyl, (optionally substitutedaryl)carbonyl, (optionally substituted heterocyclyl)carbonyl,(optionally substituted heterocyclylalkyl)carbonyl, (optionallysubstituted (C₁-C₆)-alkoxy)carbonyl, (optionally substituted(C₂-C₁₀)-alkenyloxy)carbonyl, (optionally substituted amino)carbonyl,carboxy, formyl, or hydroxy(optionally substituted)(C₁-C₆)-alkyl;

[0026] R¹⁰ is: (C₁-C₆)-alkyl, or (C₂-C₁₀)-alkenyl, and amino;

[0027] R′ is: hydrogen, (C₁-C₆)-alkyl, (C₁-C₆)-alkylcarbonyl,phosphoryl, or polyalkoxy; or

[0028] R′ and R⁶ with the atoms to which they are attached form anoptionally substituted ring; and single stereoisomers, mixtures ofstereoisomers, and the pharmaceutically acceptable salts thereof.

[0029] A preferred embodiment of this invention concerns the compoundsof Formula I where R² and R³ are (C₁-C₆)-alkyl, preferably methyl, andwithin that subset those compounds of Formula I wherein R is hydrogen.

[0030] In another embodiment, the invention concerns the compounds ofFormula I wherein R² and R³ are (C₁-C₆)-alkyl, preferably methyl, R ishydrogen, R⁵ is hydrogen, and R⁴ is optionally substituted aryl,(optionally substituted alkyl)carbonyl, (optionally substitutedaryl)carbonyl, (optionally substituted heterocyclyl)carbonyl,(optionally substituted heterocyclylalkyl)carbonyl, (optionallysubstituted alkoxy)carbonyl, (optionally substitutedalkenyloxy)carbonyl, (optionally substituted amino)carbonyl, carboxy,formyl, or hydroxy(optionally substituted)alkyl, especially wherein saidaryl is unsubstituted phenyl or substituted phenyl with one or moresubstitutents selected from alkyl, alkoxy, hydroxy, (optionallysubstituted alkoxy)carbonyl, nitro, halo, and cyano.

[0031] In another embodiment, the invention concerns the compounds ofFormula I wherein R² and R³ are (C₁-C₆)-alkyl, preferably methyl, R ishydrogen, R⁴ is hydrogen, and R⁵ is optionally substituted aryl,preferably unsubstituted phenyl or phenyl substituted with one or moresubstitutents selected from alkyl, alkoxy, hydroxy, (optionallysubstituted alkoxy)carbonyl, nitro, halo, and cyano.

[0032] In another embodiment, the invention concerns the compounds ofFormula I wherein R and R¹ form a ring, preferably R and R¹ form a furanring substituted with an unsubstituted phenyl or with a phenylsubstituted with one or more substitutents selected from alkyl, alkenyl,hydroxy, alkoxy, nitro, cyano, carboxy, carboxyester, haloalkyl, andhalo.

[0033] In another embodiment the invention concerns the novel compoundsof Formula II:

[0034] wherein:

[0035] R⁶ is: hydrogen, optionally substituted (C₁-C₆)-alkyl, optionallysubstituted (C₂-C₁₀)-alkenyl, optionally substituted aryl, (optionallysubstituted (C₁-C₆)-alkoxy)carbonyl, or halogen;

[0036] R⁷ and R⁸ are independently selected from optionally substituted(C₁-C₆)-alkyl, optionally substituted (C₂-C₁₀)-alkenyl, or optionallysubstituted (C₃-C₈)-cycloalkyl;

[0037] R⁹ is: optionally substituted aryl, (optionallysubstituted(C₁-C₆)-alkyl)carbonyl, (optionally substitutedaryl)carbonyl, (optionally substituted heterocyclyl)carbonyl,(optionally substituted heterocyclylalkyl)carbonyl, (optionallysubstituted (C₁-C₆)-alkoxy)carbonyl, (optionally substituted(C₂-C₁₀)-alkenyloxy)carbonyl, (optionally substituted amino)carbonyl,carboxy, formyl, or hydroxy(optionally substituted)(C₁-C₆)-alkyl;

[0038] R¹⁰ is: (C₁-C₆)-alkyl, or (C₂-C₁₀)-alkenyl; and

[0039] R′ is: hydrogen, (C₁-C₆)-alkyl, (C₁-C₆)-alkylcarbonyl,phosphoryl, or polyalkoxy; or

[0040] R′ and R⁶ with the atoms to which they are attached form anoptionally substituted ring; and single stereoisomers, mixtures ofstereoisomers, and the pharmaceutically acceptable salts thereof, withthe proviso that the compound is not(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone or3-amino-5-hydroxy-4,6,7-trimethyl-benzofuran-2-carboxylic acid ethylester.

[0041] In another embodiment the invention concerns the compounds ofFormula II wherein R⁷ and R⁸ are (C₁-C₆)-alkyl and R⁶ is hydrogen, inanother embodiment R¹⁰ is (C₁-C₆)-alkyl and R′ is hydrogen, in anotherembodiment R⁹ is optionally substituted aryl carbonyl, preferablyphenylcarbonyl wherein the phenyl group may be unsubstituted orsubstituted with one or more substituents selected from alkyl, alkenyl,alkoxy, hydroxy, hydroxyalkyl, haloalkyl, (optionally substitutedalkoxy)carbonyl, carboxy, nitro, halo, and cyano. In another embodimentthe invention concerns the compounds of Formula II wherein R¹⁰ is amino,R⁹ is substituted (C₁-C₆)-alkoxycarbonyl and in another embodiment thecompound is 3-amino-5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acidmethyl ester.

[0042] Certain embodiments of the invention provide novel and preferredcombinations of the substituent groups pendant from the formulae of thedifferent inventions.

[0043] In another aspect, the invention relates to pharmaceutical and/orcosmetic compositions containing a therapeutically effective amount of acompound of any of Formula I or Formula II, or a pharmaceuticallyacceptable salt thereof, admixed with at least one pharmaceuticallyacceptable excipient. Particularly preferred are those pharmaceutical orcosmetic compositions wherein a compound of Formula I or Formula II isselected from the Preferred Compounds.

[0044] Another aspect of the present invention concerns methods oftreatment for a mammal suffering from a condition characterized byoxidative stress by administering a therapeutically effective amount ofa compound represented by the Formula I or Formula II, including singlestereoisomers, mixtures of stereoisomers, and the pharmaceuticallyacceptable salts thereof.

[0045] In one embodiment, the invention relates to a method of treatmentof a cardiovascular, cerebrovascular, neurologic, inflammatory,autoimmune, and/or dermatologic condition.

[0046] In another embodiment the invention relates to a conditionselected from stroke, cerebral ischemia, myocardial infarction, chronicheart failure, retinal ischemia, post-surgical cognitive dysfunctions,peripheral neuropathy, spinal cord injury, head injury and surgicaltrauma. In another embodiment the condition involves inflammatory orautoimmune components.

[0047] In another embodiment the condition relates to a method oftreatment diseases involving mitochondrial dysfunction, particularlywith neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, Friedreich's ataxia and other cerebellar ataxias, Leber'shereditary optic neuropathy, and epilepsy with a compound of Formula Ior of Formula II., particularly with(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone or3-amino-5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methylester; or a compound of Formula I wherein R¹ is halogen, R⁴ is(optionally substituted(C₁-C6)-alkyl)carbonyl and R⁵ is hydrogen; or acompound of Formula II wherein R⁷, R⁸ and R¹⁰ are (C₁-C₆)-alkyl, and R⁹is phenylcarbonyl, wherein said phenyl group is substituted with one ormore substituents selected from alkyl, alkenyl, alkoxy, hydroxy,hydroxyalkyl, haloalkyl, (optionally substituted alkoxy)carbonyl,carboxy, nitro, halo, and cyano, or a compound of Formula II wherein R⁷and R⁸ are alkyl, R¹⁰ is amino, and R⁹ is (C₁-C₆)-alkoxycarbonyl.

[0048] In still another embodiment, the invention relates to a method oftreating stroke and other oxidative stress-related conditions that areresponsive to cellular enzyme modulation such as cerebral ischemia,myocardial infarction, chronic heart failure, and exposure to UVradiation in a mammal, by administering to a mammal in need of suchtreatment a therapeutically effective amount of a compound of any ofFormula I or Formula II, or a pharmaceutically acceptable salt thereof.

[0049] In still another aspect, the invention relates to a method ofreducing levels of C-reactive protein (CRP) associated withinflammation, including but not limited to cardiovascular inflammatorycondition, respiratory inflammatory condition, sepsis, diabetes, musclefatigue, systemic lupus erythematosis (SLE), end stage renal disease(ERSD), periodontal disease, and inflammatory skin conditions.

[0050] Particularly preferred are those methods of treatment and uses inthe manufacture of pharmaceutical and/or cosmetic compositions thereof,wherein a compound of Formula I or Formula II is selected from thePreferred Compounds.

DETAILED DESCRIPTION OF THE INVENTION

[0051] Definitions

[0052] As used in the present specification, the following words andphrases are generally intended to have the meanings as set forth below,except to the extent that the context in which they are used indicatesotherwise.

[0053] The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined below. Itwill be understood by those skilled in the art with respect to any groupcontaining one or more substituents that such groups are not intended tointroduce any substitution or substitution patterns that are stericallyimpractical and/or synthetically non-feasible.

[0054] The term “acyl” refers to the groups —C(O)—H, —C(O)-(optionallysubstituted alkyl), —C(O)-(optionally substituted cycloalkyl),—C(O)-(optionally substituted alkenyl), —C(O)-(optionally substitutedcycloalkenyl), —C(O)-(optionally substituted aryl), —C(O)-(optionallysubstituted heteroaryl) and —C(O)-(optionally substituted heterocyclyl).

[0055] The term “alkenyl” refers to a monoradical branched orunbranched, unsaturated or polyunsaturated hydrocarbon chain, havingfrom about 2 to 20 carbon atoms, more preferably about 2 to 10 carbonatoms. This term is exemplified by groups such as ethenyl, but-2-enyl,3-methyl-but-2-enyl (also referred to as “prenyl”), octa-2,6-dienyl,3,7-dimethyl-octa-2,6-dienyl (also referred to as “geranyl”), and thelike.

[0056] The term “substituted alkenyl” refers to an alkenyl group inwhich 1 or more (up to about 5, preferably up to about 3) hydrogen atomsis replaced by a substituent, for example: hydroxy, alkoxy, carboxy,cyano, halogen or nitro.

[0057] The term “alkoxy” refers to the groups —O-alkyl, —O-alkenyl,—O-cycloalkyl, —O-cycloalkenyl, and —O-alkynyl. Preferred alkoxy groupsare —O-alkyl and —O-alkenyl and include, by way of example, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, 3,7-dimethyl-octa-2,6-dienyloxyand the like.

[0058] The term “substituted alkoxy” refers to the groups—O-(substituted alkyl), —O-(substituted alkenyl), —O-(substitutedcycloalkyl), —O-(substituted cycloalkenyl), —O-(substituted alkynyl) and—O-(optionally substituted alkylene)-alkoxy. One preferred substitutedalkoxy group is “polyalkoxy” or —O-(substituted alkylene)-alkoxy, andincludes groups such as —OCH₂OCH₃, —OCH₂CH₂OCH₃, and (or PEG) groupssuch as —O(CH₂CH₂O)_(x)CH₃ and —O(CH₂CH₂O)_(x)H where x is an integer ofabout 2-20, preferably about 2-10, and more preferably about 2-5.

[0059] The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from about 1 to 20 carbonatoms, more preferably about 1 to 10 carbon atoms, and even morepreferably about 1 to 6 carbon atoms. This term is exemplified by groupssuch as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,n-hexyl, n-decyl, tetradecyl, and the like.

[0060] The term “substituted alkyl” refers to an alkyl group in which 1or more (up to about 5, preferably up to about 3) hydrogen atoms isreplaced by a substituent independently selected from the group: ═O, ═S,acyl, acyloxy, optionally substituted alkoxy, optionally substitutedamino, azido, carboxyl, (optionally substituted alkoxy)carbonyl,(optionally substituted amino)carbonyl, cyano, optionally substitutedcycloalkyl, optionally substituted cycloalkenyl, heterocyclyl, carboxy,halogen, hydroxyl, nitro, cyano, sulfanyl, sulfinyl, and sulfony, or twosubstituents with the carbon to which they are attached may form a ring.One of the preferred optional substituents for alkyl is hydroxy,exemplified by hydroxyalkyl groups, such as 2-hydroxyethyl,3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl, and the like;dihydroxyalkyl groups (glycols), such as 2,3-dihydroxypropyl,3,4-dihydroxybutyl, 2,4-dihydroxybutyl, and the like; and thosecompounds known as polyethylene glycols, polypropylene glycols andpolybutylene glycols, and the like. A preferred “substituted alkyl”wherein the substitutents form a ring is6-hydroxy-3-methyl-[1,3]oxazinan-6-yl.

[0061] The term “Alzheimer's Disease” (“AD”) refers to a progressivedisease of the human central nervous system. It is manifested bydementia in the elderly, by disorientation, loss of memory, difficultywith language, calculation, or visual-spatial skills, and by psychiatricmanifestations. It is associated with degenerating neurons in severalregions of the brain. Alzheimer's Disease is reviewed by Price, D. L. etal. (Clin. Neuropharm. 14:S9-S14 (1991)); Pollwein, P. et al. (Nucl.Acids Res. 20:63-68 (1992)); Regland, B. et al. (Med. Hypoth. 38:11-19(1992)) and Johnson, S. A. (In: Review of Biological Research in Aging,Vol. 4., Rothstein, M. (Ed.), Wiley-Liss, N.Y., 163-170 (1990)).

[0062] The term “amino” refers to the group —NH₂, —NHR, or —NRR whereeach R is independently selected from the group: optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substitutedalkenyl, optionally substituted cycloalkenyl, optionally substitutedalkynyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclyl, acyl, optionally substitutedalkoxy, carboxy and alkoxycarbonyl, and wherein RR form with thenitrogen to which they are attached a cyclic amine optionallyincorporating one or more additional heteroatoms selected from O, N andS.

[0063] The term “aryl” refers to an aromatic cyclic hydrocarbon group offrom 6 to 20 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed (fused) rings (e.g., naphthyl or anthryl). Preferredaryls include phenyl, naphthyl and the like.

[0064] The term “substituted aryl” refers to an aryl group as definedabove, which unless otherwise constrained by the definition for the arylsubstituent, is substituted with from 1 to 5 substituents, andpreferably 1 to 3 substituents, independently selected from the groupconsisting of: ═O, ═S, acyl, acyloxy, optionally substituted alkenyl,optionally substituted alkoxy, optionally substituted alkyl (such astri-halomethyl), optionally substituted alkynyl, optionally substitutedamino, optionally substituted aryl, optionally substituted aryloxy,azido, carboxyl, (optionally substituted alkoxy)carbonyl, (optionallysubstituted amino)carbonyl, cyano, optionally substituted cycloalkyl,optionally substituted cycloalkenyl, halogen, optionally substitutedheteroaryl, optionally substituted heteroaryloxy, optionally substitutedheterocyclyl, optionally substituted heterocyclooxy, hydroxyl, nitro,sulfanyl, sulfinyl, and sulfonyl. Preferred aryl substituents includeoptionally substituted alkenyl, alkyl, alkoxy, substituted amino, halo,hydroxy, alkoxycarbonyl, carboxy, cyano, nitro, phosphoryl.

[0065] The term “carbonyl” refers to the di-radical “—C(═O)—”, which isalso illustrated as “—C(O)—”.

[0066] The term “(optionally substituted alkoxy)carbonyl” refers to thegroups: —C(O)O-(optionally substituted alkyl), —C(O)O-(optionallysubstituted cycloalkyl), —C(O)O-(optionally substituted alkenyl),—C(O)O-(optionally substituted alkynyl), —C(O)O-(optionally substitutedaryl), —C(O)O-(optionally substituted heteroaryl), and—C(O)O-(optionally substituted heterocyclyl). These moieties are alsoreferred to as esters.

[0067] The term “(optionally substituted amino)carbonyl” refers to thegroup —C(O)-(optionally substituted amino). This moiety is also referredto as a primary, secondary or tertiary carboxamide, and the “substitutedamino” can be a cyclic amine.

[0068] The term “carboxy” or “carboxyl” refers to the moiety “—C(O)OH”,which is also illustrated as “—COOH”.

[0069] The term “compound of Formula I” or “compound of Formula II”, isintended to encompass the derivatives of the invention as disclosed,and/or the pharmaceutically acceptable salts of such compounds. Inaddition, the compounds employed in this invention include theindividual stereochemical isomers (arising from the selection ofsubstituent groups) and mixtures of isomers.

[0070] The term “CRP” or “C-reactive protein” refers to a biochemicalmarker of inflammation. The presence of elevated levels of CRP has beenshown to be associated with various inflammatory conditions such as forexample, cardiovascular diseases or disorders, including atrialfibrillation, unstable angina, coronary artery disease, peripheralartery disease, cardiac allograft vasculopathy (CAVD); mastitis;preeclampsia; inflammatory bowel conditions; stroke; tissue infarction;lumbosciatic; estrogen/progestin hormone replacement therapy (HRT);infection (bacterial, viral and protozoan); bacterial meningitis;trauma; surgery; biomaterial implants; smoking; obesity;neurodegenerative diseases such as, Alzheimers; infectious disease, suchas, for example, myocarditis, cardiomyopathy, acute endocarditis,pericarditis; atherosclerosis; Systemic Inflammatory Response Syndrome(SIRS)/sepsis; adult respiratory distress syndrome (ARDS); asthma;rheumatoid arthritis, osteoarthritis, systemic lupus erythematosis;Airway hyper-responsiveness (AHR); bronchial hyper-reactivity; ChronicObstructive Pulmonary disease (COPD); Congestive Heart Failure (CHF);inflammatory complications of diabetes mellitus type I and type II;metabolic syndrome; end stage renal disease (ESRD), pre-menstrualsyndrome (PMS) or muscle fatigue or inflammation; multiple organdysfunction syndrome (MODS); airway hyper-responsiveness (AHR);bronchial hyper-reactivity; aging; acute allergic reactions; gingivitisand dermal conditions. CRP has been reported as a marker for systemicinflammation Spanheimer (2001, Postgrad. Med. 109 (4) 26) and Ridkler etal (2000, N.E.J.M. 342 (12) 836-43).

[0071] The term “cycloalkyl” refers to the monovalent saturated radicalconsisting of one or more rings, which can optionally be susbstitutedwith hydroxy, cyano, lower alkyl, lower alkoxy, thioalkyl, halo,haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino,dialkylamino, aminocarbonyl, carbonylamino, aminosulfonyl, sulfonylaminoor sulfonyl, unless otherwise indicated. Examples of cycloalkyl radicalsinclude but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl,etc.

[0072] The term “epilepsy” refers to any neurological condition thatmakes people susceptible to seizures. A seizure is a change insensation, awareness, or behavior brought about by a brief electricaldisturbance in the brain. Seizures vary from a momentary disruption ofthe senses, to short periods of unconsciousness or staring spells, toconvulsions. Some people have just one type of seizure. Others have morethan one type. Although they look different, all seizures are caused bythe same thing: a sudden change in how the cells of the brain sendelectrical signals to each other.

[0073] The term “Friedreich's ataxia” refers to an autosomal recessivemulti-system degenerative disorder that results in progressive damage tothe nervous system and causes symptoms ranging from muscle weakness andspeech problems to heart disease. Ataxia results from the degenerationof nerve tissue in the spinal cord and of nerves that control musclemovement in the arms and legs. Symptoms usually begin between the agesof 5 and 15 but can appear as early as 18 months or as late as 30 yearsof age. The first symptom is usually difficulty in walking. The ataxiagradually worsens and slowly spreads to the arms and then the trunk.Foot deformities such as clubfoot, flexion (involuntary bending) of thetoes, hammer toes, or foot inversion (turning in) may be early signs.Rapid, rhythmic, involuntary movements of the eyeball are common. Mostpeople with Friedreich's ataxia develop scoliosis (a curving of thespine to one side), which, if severe, may impair breathing. Othersymptoms include chest pain, shortness of breath, and heartpalpitations.

[0074] The term “halo” or “halogen” refers to fluoro, chloro, bromo, andiodo.

[0075] The term “heteroaryl” refers to an aromatic cyclic hydrocarbongroup having about 1 to 40 (preferably from about 3 to 15) carbon atomsand about 1 to 10 hetero atoms (preferably about 1 to 4 heteroatoms,selected from nitrogen, sulfur, phosphorus, and/or oxygen) within atleast one ring. Such heteroaryl groups can have a single ring (e.g.,pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl orbenzothienyl). Preferred heteroaryls include pyridyl, [2,2′]bipyridinyl,pyrrolyl and furyl.

[0076] The terms “heterocycle”, “heterocyclic”, “heterocyclo”, and“heterocyclyl” refer to a monoradical, saturated or unsaturated,non-aromatic cyclic hydrocarbon group having about 1 to 40 (preferablyfrom about 3 to 15) carbon atoms and about 1 to 10 hetero atoms(preferably about 1 to 4 heteroatoms, selected from nitrogen, sulfur,phosphorus, and/or oxygen) within the ring. Such heterocyclic groups canhave a single ring or multiple condensed rings. Preferred heterocyclicsinclude morpholino, piperidinyl, 1,3-oxazinane, and the like.

[0077] The terms “substituted heterocycle”, “substituted heterocyclic”,“substituted heterocyclyl” and “substituted heterocyclo” refer to aheterocyclyc group as defined above, which unless otherwise constrainedby the definition for the heterocycle, is substituted with from 1 to 5substituents, and preferably 1 to 3 substituents, independently selectedfrom the group consisting of: ═O, ═S, acyl, acyloxy, optionallysubstituted alkenyl, optionally substituted alkoxy, optionallysubstituted alkyl (such as tri-halomethyl), optionally substitutedalkynyl, optionally substituted amino, optionally substituted aryl,optionally substituted aryloxy, azido, carboxyl, (optionally substitutedalkoxy)carbonyl, (optionally substituted amino)carbonyl, cyano,optionally substituted cycloalkyl, optionally substituted cycloalkenyl,halogen, optionally substituted heteroaryl, optionally substitutedheteroaryloxy, optionally substituted heterocyclyl, optionallysubstituted heterocyclooxy, hydroxyl, nitro, sulfanyl, sulfinyl, andsulfonyl.

[0078] The term “heterocycloalkyl” refers to the moiety“-alkylene-heterocycle” each having the meaning as defined herein.

[0079] The term “substituted heterocycloalkyl” refers to the moiety“—(optionally substituted aklylene)—(optionally substitutedheterocycle)”, each having the meaning as defined herein.

[0080] The term “hydroxy (optionally substituted) alkyl” refers to thegroup -(optionally substituted alkyl)—OH, wherein 13 (optionallysubstituted alkyl)—is an unsubstituted or a substituted alkyl group asdescribed herein. This term is exemplified by the groups2-hydroxymethyl, 1-hydroxy-2-morpholin-4-yl-ethyl, and6-hydroxy-3-methyl-[1,3]oxazinan-6-yl.

[0081] The term “inflammation”, “inflammatory conditions”, or“inflammation conditions” includes but is not limited to muscle fatigue,osteoarthritis, rheumatoid arthritis, inflammatory bowel syndrome ordisorder, skin inflammation, such as atopic dermatitis, contactdermatitis, allergic dermatitis, xerosis, eczema, rosacea, seborrhea,psoriasis, atherosclerosis, thermal and radiation burns, acne, oilyskin, wrinkles, excessive cellulite, excessive pore size, intrinsic skinaging, photo aging, photo damage, harmful UV damage, keratinizationabnormalities, irritation including retinoid induced irritation,hirsutism, alopecia, dyspigmentation, inflammation due to wounds,scarring or stretch marks, loss of elasticity, skin atrophy andgingivitis.

[0082] The term “ischemia” refers to deficiency of blood to an organ ortissue due to functional constriction or actual obstruction of a bloodvessel. Cerebral ischemia, also known as stroke, usually results fromthe interruption or reduction of blood and oxygen to the blood vesselsof the brain; more rarely this may be the result of an hemorrhage. Signsof stroke include paralysis, slurred speech, general confusion,impairment of gait, cortical sensory loss over toes, foot and leg, andurinary incontinence, to name just a few. Many types of heart diseaseincluding cardiac arrhythmias or diseases due to cardiac structuralabnormalities may produce cerebral emboli. Atrial fibrillation from anycause, including rheumatic valvular disease, may result in emboli beingproduced which can migrate into the arteries of the brain. Emboliformation and migration can occur as a result of atheroscleroticcardiovascular disease and myocardial infarction. Emboli formation isalso a definite risk for intracardiac surgery and prosthetic valvereplacement. Heart bypass surgery and angioplasty can result in theformation of microemboli which can migrate into the arteries of thebrain and cause a series of occlusions in a number of arteries,resulting in mental impairment. Cerebral embolism is also the principalcomplication in the transplant of artificial hearts. Furthermore, theoverall risk of stroke after any type of general surgery is 0.2 to 1percent. The vegetations of acute and subacute bacterial endocarditiscan give rise to emboli which can occlude a major intracranial artery.Populations at risk of ischemia include but are not limited to patientsscheduled for coronary arterial bypass graft surgery (CABG), patients atrisk for postoperative complications, patients with subarachnoidhemorrhage (SAH), patients with a first or second ischemic stroke,patients with acute ischemic stroke, patients undergoing cardiopulmonaryresuscitation (CPR), patients with temporary lobectomy, patients withdominant hemisphere resection, patients receiving prophylactic brainradiation, patients with closed head trauma with neurological loss,patients with microvascular multi-infarct dementia, patients withhomozygous and heterozygous MELAS (Mitochondrial myopathy,encephalopathy, lactacidosis, stroke); patients with atherosclerotic orprogressive supranuclear palsy disease, patients with symptomatic andasymptomatic Huntington's disease, patients with neonatal asphyxia,patients with meningitis or encephalitis, patients with post herpeticneuropathy, patients with intermittent claudication, patients withspinal cord injury, patients with Huntington's disease, AmyotrophicLateral Sclerosis (ALS) or Friedreich's ataxia, patients with diabeticneuropathy or patients with a disease associated with a hypercoagulablestate secondary to systemic disease, carcinoma, vasoconstriction(including reversible cerebral vasoconstriction, e.g. migraine , trauma,idiopathy), or venous conditions (including dehydration, pulmonaryembolism, pericranial infection, postpartum and postoperative states andsystem cancer).

[0083] The term “mitochondrial associated diseases” refers todegenerative diseases that may be caused by or associated with eitherdirect or indirect alterations in mitochondrial dysfunctions. Theseinclude Alzheimer's disease, diabetes mellitus, Parkinson's disease,neuronal and cardiac ischemia, Huntington's disease, spinal bulbarmuscular atrophy, Macado-Joseph disease, Dentatorubral-PallidoluysianAtrophy (DRPLA), Friedreich's ataxia (FRDA) and other spinocerebellarataxias, dystonia, Leber's hereditary optic neuropathy (LHON),schizophrenia, MELAS and MERFF.

[0084] The term “neurodegeneration” refers to a progressive death anddisappearance of a population of nerve cells occurring in a mannercharacteristic of a particular disease state and leading to brain orother neuronal damage. It is the process of cell destruction resultingfrom primary destructive events, or secondary delayed and progressivedestructive mechanisms that are invoked by cells due to the occurrenceof the primary destructive event. Primary destructive events includedisease processes or physical injury or insult, including stroke,multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease,Parkinson's disease, epilepsy, cerebral ischemia, and physical traumasuch as crush or compression injury in the CNS, including a crush orcompression injury of the brain, spinal cord, nerves or retina, or anyacute injury or insult. Secondary destructive mechanisms include anymechanism that leads to the generation and release of neurotoxicmolecules, including apoptosis, depletion of cellular energy storesbecause of changes in mitochondrial membrane permeability, release orfailure to reuptake excessive glutamate, reperfusion injury, andactivity of cytokines and inflammation.

[0085] The term “neurodegenerative condition” refers to a primarydestructive event or secondary destructive mechanism resulting inneurodegeneration as defined herein.

[0086] The term “neuroprotection” refers to a therapeutic strategy forslowing or preventing the irreversible loss of neurons due toneurodegeneration after a primary destructive event, whether theneurodegeneration loss is due to disease mechanisms associated with theprimary destructive event or to secondary destructive mechanisms.

[0087] The term “Parkinson's disease” (“PD”) refers to aneurodegenerative disease especially affecting the dopaminergic neuronsof the substantia nigra—pars compacta—and its nigrostriatal projectionsin nearly one million Americans. The symptomatic manifestations aremotor disorders such as tremor, muscular rigidity, bradykinesia, poorbalance and walking problems. Secondary symptoms include depression,sleep disturbances, dizziness, stooped posture, constipation, dementia,and problems with speech, breathing, swallowing, and sexual functionsymptoms. Due to their mimicry of effects of Parkinson's Disease,treatment of animals with methamphetamine or MPTP has been used togenerate models for Parkinson's Disease. These animal models have beenused to evaluate the efficacy of various therapies for Parkinson'sDisease.

[0088] The terms “pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient” include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0089] The term “pharmaceutically acceptable salt” refers to salts whichretain the biological effectiveness and properties of the compounds ofthis invention and which are not biologically or otherwise undesirable.In many cases, the compounds of this invention are capable of formingacid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto. Pharmaceutically acceptablebase addition salts can be prepared from inorganic and organic bases.Salts derived from inorganic bases, include by way of example only,sodium, potassium, lithium, ammonium, calcium and magnesium salts. Saltsderived from organic bases include, but are not limited to, salts ofprimary, secondary and tertiary amines, such as alkyl amines, dialkylamines, trialkyl amines, substituted alkyl amines, di(substituted alkyl)amines, tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines,trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl)amines, tri(substituted alkenyl) amines, cycloalkyl amines,di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkylamines, disubstituted cycloalkyl amine, trisubstituted cycloalkylamines, cycloalkenyl amines, di(cycloalkenyl) amines, tri(cycloalkenyl)amines, substituted cycloalkenyl amines, disubstituted cycloalkenylamine, trisubstituted cycloalkenyl amines, aryl amines, diaryl amines,triaryl amines, heteroaryl amines, diheteroaryl amines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclicamines, mixed di- and tri-amines where at least two of the substituentson the amine are different and are selected from the group consisting ofalkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,heteroaryl, heterocyclic, and the like. Also included are amines wherethe two or three substituents, together with the amino nitrogen, form aheterocyclic or heteroaryl group.

[0090] Pharmaceutically acceptable acid addition salts may be preparedfrom inorganic and organic acids. Salts derived from inorganic acidsinclude hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

[0091] The term “phosphoryl” refers to the group —P(O)(OR″)₂, where R″is independently selected from hydrogen or alkyl and aryl, which groupis sometimes also referred to as “phosphono” or as a “phosphate” or“phosphonic acid.”

[0092] The term “therapeutically effective amount” refers to that amountof a compound of this invention that is sufficient to effect treatment,as defined below, when administered to a mammal in need of suchtreatment. The therapeutically effective amount will vary depending uponthe subject and disease condition being treated, the weight and age ofthe subject, the severity of the disease condition, the particularcompound chosen, the dosing regimen to be followed, timing ofadministration, the manner of administration and the like, all of whichcan readily be determined by one of ordinary skill in the art.

[0093] The term “treatment” or “treating” means any treatment of adisease or disorder in a mammal, including:

[0094] preventing or protecting against the disease or disorder, thatis, causing the clinical symptoms not to develop;

[0095] inhibiting the disease or disorder, that is, arresting orsuppressing the development of clinical symptoms; and/or

[0096] relieving the disease or disorder that is causing the regressionof clinical symptoms.

[0097] It will be understood by those skilled in the art that in humanmedicine, it is not always possible to distinguish between “preventing”and “suppressing” since the ultimate inductive event or events may beunknown, latent, or the patient is not ascertained until well after theoccurrence of the event or events.

[0098] Therefore, as used herein the term “prophylaxis” is intended asan element of “treatment” to encompass both “preventing” and“suppressing” as defined herein. The term “protection,” as used herein,is meant to include “prophylaxis.”

[0099] Nomenclature

[0100] In general the nomenclature used in this Application is based onAutonom™ v.2.1, a Beilstein Institute computerized system for thegeneration of IUPAC systematic nomenclature.

[0101] The compounds of the present invention are named and numbered asdescribed below.

[0102] Formula Ia represents the compound according to Formula I whereR, R¹ and R⁵ are hydrogen, R² and R³ are methyl, and R⁴ isp-nitro-phenyl, and can be named6,7-dimethyl-2-(4-nitro-phenyl)-benzofuran-5-ol.

[0103] Formula Ib represents the compound according to Formula I whereR, R¹, and R⁴ are hydrogen, R² and R³ are methyl, and R⁵ is phenyl, andcan be named 6,7-dimethyl-3-phenyl-benzofuran-5-ol.

Synthesis of the Compounds of the Invention

[0104] Synthetic Reaction Parameters

[0105] The terms “solvent”, “inert organic solvent” or “inert solvent”mean a solvent inert under the conditions of the reaction beingdescribed in conjunction therewith. Solvents employed in synthesis ofthe compounds of the invention include, for example, methanol (“MeOH”),acetone, water, acetonitrile, 1,4-dioxane, dimethylformamide (“DMF”),benzene, toluene, xylene, tetrahydrofuran (“THF”), chloroform, methylenechloride (or dichloromethane, (“DCM”)), diethyl ether, pyridine and thelike, as well as mixtures thereof. Unless specified to the contrary, thesolvents used in the reactions of the present invention are inertorganic solvents.

[0106] The term “q.s.” means adding a quantity sufficient to achieve astated function, e.g., to bring a solution to the desired volume (i.e.,100%).

[0107] Unless specified to the contrary, the reactions described hereintake place at atmospheric pressure within a temperature range from −10°C. to 110° C. (preferably from 0° C. to 40° C.; most perferably at“room” or “ambient” temperature, e.g., 20° C.). Further, unlessotherwise specified, the reaction times and conditions are intended tobe approximate, e.g., taking place at about atmospheric pressure withina temperature range of about −10° C. to about 110° C. (preferably fromabout 0° C. to about 40° C.; most preferably at about “room” or“ambient” temperature, e.g., approximately 20° C.) over a period ofabout 1 to about 10 hours (preferably about 5 hours). Parameters givenin the Examples are intended to be specific, not approximate.

[0108] Isolation and purification of the compounds and intermediatesdescribed herein can be effected, if desired, by any suitable separationor purification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, or a combination of these procedures.Specific illustrations of suitable separation and isolation procedurescan be had by reference to the examples hereinbelow. However, otherequivalent separation or isolation procedures can, of course, also beused.

[0109] Starting Materials

[0110] The starting compounds , e.g., 2,3-dimethylhydroquinone, arecommercially available, e.g. from Aldrich Chemical Company, Milwaukee,Wiss., or may be readily prepared by those skilled in the art usingcommonly employed methodology.

[0111] Referring to Reaction Scheme 1, a 2,3-substituted hydroquinone ofFormula 101 is treated with one equivalent of a2-halo-1-substituted-ethanone, preferably2-bromo-1-substituted-ethanone, most preferably 2-bromo-1-substitutedphenyl ethanone, in the presence of a base such as sodium carbonate,potassium carbonate or cesium carbonate in a solvent such as acetone,THF, or DMF, to give2-(4-hydroxy-2,3-substituted-phenoxy)-1-substituted-ethanone of Formula102. Compound of Formula 102 can subsequently be treated with an acidsuch as polyphosphoric acid, hydrochloric acid, or sulfuric acid in aninert solvent such as toluene or xylene, to cyclise into a benzofurancompound of Formula 103.

[0112] Similarly treating a 2,3-substituted hydroquinone of Formula 102with 2 or more equivalents of a 2-halo-1-substituted ethanone in thepresence of a base, followed by cyclization in the presence of an acid,can give a compound of Formula 105.

[0113] Referring to Scheme 2, a compound of Formula 201 wherein “Prot”is a hydroxy protecting group, preferably a tetrahydropyranyl group,when reacted with a 2-halo-1-substituted ethanone, preferably a2-bromo-1-substituted ethanone in the presence of a base such as sodiumcarbonate, potassium carbonate, or cesium carbonate in a solvent such asacetone, THF, or DMF, followed by cyclization in the presence of an acidas described in reaction scheme 1, can give a compound of Formula 202.

Preferred Compounds

[0114] The following combinations and permutations of substituent groups(sub-grouped, respectively, in increasing order of preference) definecompounds that are preferred as compositions of matter and compounds foruse in the methods and pharmaceutical and cosmetic compositionsaccording to the invention.

[0115] The compounds of any of Formula I where R² and R³ are optionallysubstituted alkyl, particularly those wherein R² and R³ are methyl.

[0116] Preferably those where R¹ is hydrogen or halogen

[0117] Especially those where R is hydrogen.

[0118] The compounds of any of Formula I where R¹ is hydrogen or halo

[0119] Preferably those where R¹ is hydrogen

[0120] The compounds of any of Formula I where R is hydrogen, alkyl,acyl, phosphoryl or polyalkoxy, preferably hydrogen, and R¹ is hydrogen

[0121] 1. Particularly those wherein R² and R³ are methyl

[0122] Especially those of Formula I where R⁵ is optionally substitutedaryl, wherein the substitutents are chosen from alkyl, alkoxy, hydroxy,(optionally substituted alkoxy)carbonyl, nitro, halo, and cyano

[0123] Particularly those where R⁵ is optionally substituted phenyl.

[0124] Particularly those where R⁵ is optionally substituted phenyl andR⁴ is hydrogen

[0125] Especially those of Formula I where R⁴ is optionally substitutedaryl

[0126] Particularly those where R⁴ is optionally substituted phenyl

[0127] Preferably those where R⁴ is para-substituted phenyl, wherein thesubstitutents are chosen from alkyl, alkoxy, hydroxy, (optionallysubstituted alkoxy)carbonyl, nitro, halo, and cyano

[0128] Preferably those where R⁴ is 4-nitrophenyl, 4-cyanophenyl and R⁵is hydrogen.

[0129] Especially those of Formula I where R⁴ is formyl, (optionallysubstituted alkyl)carbonyl, (optionally substituted aryl)carbonyl,(optionally substituted heterocyclyl)carbonyl, (optionally substitutedheterocyclylalkyl)carbonyl

[0130] Particularly those where R⁴ is (optionally substitutedaryl)carbonyl

[0131] Particularly those where R⁴ is (optionally substitutedalkyl)carbonyl

[0132] Particularly those where R⁴ is alkylcarbonyl optionallysubstituted with halogen, hydroxy or heterocyclyl, especiallysubstituted with morpholin-1-yl

[0133] Particularly those where R⁴ is formyl

[0134] Preferably those where R⁴ is selected from formyl,phenylcarbonyl, bromoacetyl, morpholin-1-yl-acetyl, and acetyl; and R⁵is hydrogen

[0135] Especially those wherein R⁴ is (optionally substitutedalkoxy)carbonyl, (optionally substituted alkenyloxy)carbonyl,(optionally substituted amino)carbonyl, carboxy, or hydroxy(optionallysubstituted)alkyl;

[0136] Particularly those where R⁴ is (optionally substitutedalkoxy)carbonyl, preferably those where the alkoxy is polyalkoxy, or(optionally substituted alkenyloxy) carbonyl, preferably those where thealkenyloxy is geranyloxy.

[0137] Particularly those where R⁴ is (optionally substitutedamino)carbonyl, preferably those wherein the amino is substituted withoptionally substituted alkyl, especially hydroxyalkyl, or wherein theamino is a cyclic amine.

[0138] Preferably those where R⁴ is morpholin-1-yl-carbonyl;bis-(2-hydroxy-ethyl)-amide, 2-hydroxy-ethyl-amide, carboxylic acid;carboxylic acid methyl ester; carboxylic acid3,7-dimethyl-octa-2,6-dienyl ester; [2-(2-methoxy-ethoxy)-ethoxy]ethylester; and R⁵ is hydrogen.

[0139] Especially those where R⁴ are hydroxy (optionally substituted)alkyl

[0140] Particularly where R⁴ is hydroxymethyl,1-hydroxy-2-morpholiny-4-yl ethyl or6-hydroxy-3-methyl-[1,3]oxazinan-6-yl.

[0141] The compounds of any of Formula I wherein R and R¹ with the atomsto which they are attached form an optionally substituted ring

[0142] Especially those wherein R and R¹ with the atoms to which theyare attached form a furan ring substituted with an unsubstituted phenylring or with a substituted phenyl ring, wherein one or more substituentsare independently selected from alkyl, alkenyl, halo alkyl, hydroxy,alkoxy, carboxy, ester, haloalkyl, and halo.

[0143] The compounds preferred for use in the invention include thefollowing, as well as their stereoisomers, salts, and mixtures thereof(as appropriate):

[0144] (5-Hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone;

[0145](5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-morpholin-4-yl-methanone;

[0146] 1-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone;

[0147] Acetic acid 2-(2-bromo-acetyl)-6,7-dimethyl-benzofuran-5-ylester;

[0148]2-(1-Hydroxy-2-morpholin-4-yl-ethyl)-6,7-dimethyl-benzofuran-5-ol;

[0149] Acetic acid6,7-dimethyl-2-(2-morpholiin-1-yl-acetyl)-benzofuran-5-yl ester;

[0150] Acetic acid2-(6-hydroxy-3-methyl-[1,3]oxazinan-6-yl)-6,7-dimethyl-benzofuran-5-ylester;

[0151]1-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-2-morpholin-4-yl-ethanone;

[0152] 1-(4-Bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone;

[0153] 2-Hydroxymethyl-6,7-dimethyl-benzofuran-5-ol;

[0154] 6,7-Dimethyl-3-phenyl-benzofuran-5-ol;

[0155] 6,7-Dimethyl-2-(4-nitro-phenyl)-benzofuran-5-ol;

[0156] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde;

[0157] 4-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-benzonitrile;

[0158] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid;

[0159] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester;

[0160] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid3,7-dimethyl-octa-2,6-dienyl ester;

[0161] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acidbis-(2-hydroxy-ethyl)-amide;

[0162] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester;

[0163] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid(2-hydroxy-ethyl)-amide;

[0164] 3-(4-Methoxy-phenyl)-6,7-dimethyl-benzofuran-5-ol;

[0165] 3-(4-Chloro-phenyl)-6,7-dimethyl-benzofuran-5-ol;

[0166] 3-(4-Fluoro-phenyl)-6,7-dimethyl-benzofuran-5-ol;

[0167] 4,5-Dimethyl-1,8-diphenyl-benzo[1,2-b;4,3-b′]difuran;

[0168]1,8-Bis-(4-fluoro-phenyl)-4,5-dimethyl-benzo[1,2-b;4,3-b′]difuran;

[0169]1,8-Bis-(4-methoxy-phenyl)-4,5-dimethyl-benzo[1,2-b;4,3-b′]difuran; and.

[0170] 3-Amino-5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acidmethyl ester.

Utility, Testing and Administration

[0171] General Utility

[0172] Compounds, compositions, formulations and methods of the presentinvention are useful for the treatment of disorders characterized bydefective mitochondrial activity. In particular compounds of the presentinvention can be used in the treatment of diseases such as Alzheimer'sdisease, drug-induced Parkinsonism or Parkinson's disease, Friedreich'sataxia and other ataxias, Leber's hereditary optic neuropathy, andepilepsy.

[0173] Compounds, compositions/formulations and methods of the presentinvention are useful in treating a number of disorders, particularlythose characterized by oxidative stress and/or inflammation. Inparticular, compounds of the present invention can be used in thetreatment of cerebral ischemia (“stroke”), myocardial ischemia(myocardial infarction and other forms of heart disease), diabetes,renal disease, pre-menstrual syndrome, asthma, cardiopulmonaryinflammatory disorders, chronic heart failure, rheumatoid arthritis,muscle fatigue, intermittent claudication and for the preservation ofallograft tissue for transplantation.

[0174] The compounds of the present invention have also shown use inreducing C-reactive protein (CRP) associated with inflammation and/orinflammatory conditions including cardiovascular diseases or disorders,such as atrial fibrillation, unstable angina, coronary artery disease,peripheral artery disease, cardiac allograft vasculopathy (CAVD),mastitis, preeclampsia, inflammatory bowel conditions, stroke, tissueinfarction, lumbosciatic, estrogen/progestin hormone replacement therapy(HRT); infection (bacterial, viral and protozoan), bacterial meningitis,trauma, surgery, biomaterial implants, smoking, obesity,neurodegenerative diseases such as Alzheimer's, infectious disease suchas for example myocarditis, cardiomyopathy, acute endocarditis orpericarditis, atherosclerosis, systemic inflammatory response(SIRS)/sepsis, adult respirator distress syndrome (ARDS), asthma,rheumatoid arthritis, osteoarthritis, systemic lupus erythematosis,airway hyperresponsiveness (AHR), bronchial hyper-reactivity, chronicobstructive pulmonary disease (COPD), congestive heart failure (CHF),inflammatory complications of diabetes mellitus type I and II, metabolicsyndrome, end stage renal disease (ESRD), pre-menstrual syndrome (PMS)or muscle fatigue or inflammation, multiple organ dysfunction syndrome(MODS), airway hyper-responsiveness (AHR), bronchia hyper-reactivity,aging, acute allergic reactions, periodontal disease, such as gingivitisand dermal conditions including inflammatory skin conditions.

[0175] Testing

[0176] This section describes how compositions incorporatingcompositions of the present invention are selected, using in vitroand/or in vivo animal models, for example, and used as therapeuticinterventions in three exemplary indications, i.e., stroke, chronicheart failure and myocardial infarction.

[0177] The effectiveness of a compound of this invention as amitochondria protecting agent or neuroprotecting agent may be determinedby assays such as the MPP+ Cell Death Assay, the MPTP assay or theAmyloid Assay.

[0178] Insults to the brain that disrupt its blood supply, as inischemia, or its oxygen supply, as in hypoxia (low oxygen) or anoxia (nooxygen), rapidly cause neuronal imbalance leading to cell death (Flynn,C. J., et al., 1989, in G. Siegel et al., (Eds), Basic Neurochemistry,Raven Press, NY). Investigations into the cellular and molecularmechanisms that lead to neuronal damage and inflammation associated withvarious types of brain ischemia can be carried out using in vitro modelsystems, such as primary cell cultures, that retain the metaboliccharacteristics of neurons in vivo. The use of such cell-based modelshas led to advances in identification of biochemical mechanisms leadingto neuronal death in conditions such as anoxia, hypoglycemia,excitotoxicity, and exposure to reactive oxygen species. Neuronal celllines such as the pheochromocytoma cell line, PC12, are also usefulmodels for studying the effects of oxidative stress on the structure andfunction of neuron-specific proteins that are expressed in the celllines. As many neuronal cell lines do not express all the properties ofgenuine neurons, primary neuronal cultures are now widely used as invitro models in which to discern the processes that occur in intactbrain.

[0179] In vitro models of ischemia approximate oxygen and glucosedeprivation that mimic in vivo conditions, for example, by placingneuronal cultures into large anaerobic or hypoxic chambers andexchanging culture medium with de-oxygenated and defined ioniccomposition media. The toxic overstimulation of neuronal glutamatereceptors, especially N-methyl-D-aspartate (NMDA) receptors, contributesto hypoxic-ischemic neuronal injury (Choi, D. M., 1988, Neuron 1:623-634), ischemic induction of reactive oxygen species (ROS) (Watson,B. D., et al., 1988, Ann NY Acad Sci., 59: 269-281), excessive calciuminflux (Grotta, J. C., 1988, Stroke 19: 447-454), arachidonic acidincrease (Siesjo, B. K., 1981, J. Cereb. Blood Flow Metab. 1: 155-186)and DNA damage (MacManus, J. P., et al., 1993, Neurosci. Lett., 164:89-92), each causing a cascade of neurodegeneration.

[0180] Primary embryonic hippocampal neuronal cells are widelyrecognized as useful in models of neuronal function. The hippocampus isa source of a relatively homogenous population of neurons withwell-characterized properties typical of central nervous system (CNS)neurons in general. Pyramidal neurons, the principal cell type in thehippocampus, have been estimated to account for 85% to 90% of the totalneuronal population (Banker and Goslin, 1998, Culturing Nerve Cells,2^(nd) edition. The MIT Press, Cambridge, Mass.). The hippocampus alsoexhibits a remarkable capacity for activity-dependent changes insynaptic function, such as long-term potentiation (Hawkins R D, Kandel ER, Siegelbaum S A. (1993) Learning to modulate transmitter release:themes and variations in synaptic plasticity [review], Ann. RevNeurosci. 16:625-665.).

[0181] In experiments carried out in support of the present inventionaccording to methods detailed in the Examples, anoxia/ischemia wasinduced in primary cultures of hippocampal neuronal cells, and compoundswere tested for their ability to prevent cell death. Compounds found tohave activity in such in vitro assays are then further tested in one ormore animal models of cerebral ischemia (“stroke”), such as the middlecerebral artery occlusion (MCAO) model in rats.

[0182] Briefly, primary cultures of hippocampal neurons are used to testcompounds for activity in neuronal protection. Hippocampal cultures aretypically prepared from 18- to 19-day fetal rats. At this age, thegeneration of pyramidal neurons, which begins in the rat at about E15,is essentially complete. The brain tissue at this stage is relativelyeasy to dissociate, the meninges are removed readily, and the number ofglial cells still is relatively modest (Park L C, Calingasan N Y, UchidaK, Zhang H, Gibson G E. Metabolic impairment elicits brain celltype-selective changes in oxidative stress and cell death in culture. JNeurochem 74(1):114-124, (2000)).

[0183] In order to evaluate the activity of compounds of the presentinvention, a test compound is assessed for its ability to protect cellsagainst one or more standard stressors, including hypoxia, as detailedin the Examples. In general, desirable therapeutic compound candidatesare effective in this model at concentrations less than about 1 mM andeven more preferably, less than about 100 μM. By effective, it is meantthat such compounds protect at least 20%, preferably 30%, morepreferably 40% and even more preferably 50% or more of the cells testedfrom stressor-induced death. By way of example, compounds that areeffective in providing protection over a concentration a range of about1 to 1000 μM would be expected to provide neuroprotection in vivo. Sinceprecise values may vary depending upon the specific conditions underwhich the neuroprotective cell assay is carried out, it is the intent ofthe present disclosure to provide the foregoing criteria as guidance inthe form of a benchmark against which to compare subsequently testedcompounds, rather than to provide absolute concentrations at which thecompounds of the present invention are considered to be effective.Typically, compounds that are found to be neuroprotective in such invitro cell systems are then further tested in an in vivo animal model ofneuroprotection, such as the rat middle cerebral artery occlusion modeldescribed below, or other appropriate models such as are well known inthe art.

[0184] Cerebral ischemic insults are modeled in animals by occludingvessels to, or within, the cranium (Molinari, G. F., 1986, in H. J. M.Barnett, et al., (Eds) Stroke: Pathophysiology, Diagnosis andManagement, Vol. 1, Churchill Livingstone, N.Y.). The rat middlecerebral artery occlusion (MCAO) model is one of the most widely usedtechniques to induce transient focal cerebral ischemia approximatingcerebral ischemic damage in humans, e.g., those who suffer from astroke. The middle cerebral artery used as the ischemic trigger in thismodel is the most affected vessel in human stroke. The model alsoentails a period of reperfusion, which typically occurs in human strokevictims. MCAO involving a two-hour occlusion has been found to producethe maximum size of cortical infarction obtainable without increasedmortality at twenty-four hours.

[0185] Briefly, a nylon filament is implanted into the right carotidartery of the rat. To effect occlusion, the rat is anesthetized, and thefilament is advanced into the internal carotid artery 18-20 mm from thepoint of bifurcation of internal and external arteries and a suture istightly ligated around the filament for a period of two hours. Two hourspost occlusion, animals are re-anesthetized, and the filament isremoved, to allow reperfusion for the remainder of the experiment. Testdrugs can be administered any time during this process—before, during orafter occlusion, and can be administered by one or more of a variety ofmeans, including but not limited to intracerebroventricular (ICV)infusion, intravenous (IV) infusion, intraperitoneal (IP)administration, as well as enteral administration (e.g., gavage).Animals are maintained normothermic during the experiment, as describedin the Examples. At a pre-determined time following occlusion andreperfusion, animals are sacrificed and their brains are removed andprocessed for assessment of damage as measured by infarct volume. Ingeneral, compounds are considered to have activity in this model, ifthey provide a significant reduction in total infarct volume at a dosethat is less than about 10 mg/kg, preferably less than 1 mg/kg, morepreferably less than 100 μg/kg and even more preferably less than about1 μg/kg, when administered ICV or IV. By significant reduction of totalinfarct volume is meant a reduction of at least 20%, preferably at least30%, more preferably at least 40%, and even more preferably about 50%,compared to control values.

[0186] Further validation of efficacy in neuroprotection can be assessedin functional tests, such as the grip strength test or the rotorod test.Animals treated with compounds that show neuroprotection maintain theirpre-MCAO grip strength values after MCAO, as compared to untreatedanimals, which showed a significant reduction in grip strength,indicating loss of sensorimotor function. Likewise, animals treated withcompounds that show neuroprotection also maintained their pre-MCAOrotorod activity scores after MCAO, as compared to untreated animals,which showed a significant reduction in rotorod scores, indicating lossof sensorimotor function at higher brain levels.

[0187] Similarly, primary cultures of myocytes can be used to testcompounds in vitro for ability to provide protection against heartdamage, resulting for example from myocardial ischemia or congestiveheart failure. Preparation of myocardiocytes from neonatal rats isdescribed in the Examples. Such cells are typically used to studymolecular models of myocardial ischemia (Webster, K A, Discher, D J &Bishopric, N H. J. Mol. Cell Cardiol. 27:453-458 (1995); Camilleri, L,Moins, N, Papon, J, Maublant, J, Bailly, P, de Riberolles, C & Veyre, A.Cell Biol. & Toxicol. 13:435-444 (1997); Bielawska, A E, Shapiro, J P,Jiang, L, Melkonyan, H S, Piot, C, Wolfe, C L, Tomei, L D, Hannun, Y A &Umansky, S R. Am. J. Pathol. 151:1257-1263 (1997)) and are thereforeaccepted as indicative of myoprotective activity. Exemplary stressorassays for this purpose are provided in the Examples. For example,cardiomyocytes in culture exhibit contractile (“beating”) activity; eachcardiomyocyte contraction is associated with a rise in intracellularcalcium termed a “calcium transient”. These calcium transients can bemeasured using Fluo-4, a fluorescent dye which exhibits largefluorescence intensity increases upon the binding of calcium. This assayis cell-based and tests the ability of potential cytoprotectantmolecules to guard against ischemic damage and allow the cells tomaintain their contractile function.

[0188] Further validation of compounds can be carried out in a wholeorgan assay, such as the isolated heart (Langendorff) model of cardiacfunction. Similarly, compounds can be further validated in additionalanimal models of disease (e.g., diabetes, renal failure, asthma, musclefatigue, inflammation), such as are well known in the art.

[0189] This section describes how compositions incorporatingcompositions of the present invention are selected, using in vitro andin vivo animal models and used as therapeutic interventions indermatological indications. A number of cell screening assays formediators of inflammatory response are well known in the art. Suchmediators include but are not limited to inflammatory cytokines,interleukin-1 beta, and tumor necrosis factor alpha (TNF-alpha). Othermolecules have been reported for use as markers of inflammation,including for example C-reactive protein (CRP), certain adhesionmolecules, and chemical mediators such as leukotriene, thromboxane andisoprostane.

[0190] In vitro evaluation of anti-inflammatory activity can bedetermined by well-characterized assays such as the E-selectin (ELAM)production assay or the CRP assay, and in vivo evaluation can bedetermined by the carrageenan-induced paw edema assay, as exemplified inExamples. The ELAM assay measures activity of test compounds in reducingexpression of ELAM in activated endothelial cells. Briefly, endothelialcells are created by adding known activators such aslipopolysaccharides, TNF, or IL-1.beta., alone or in some combination.Activated cells produce ELAM, which can be measured using, for example,an E-selecting monoclonal antibody-based ELISA assay. In studies carriedout in support of the present invention, ELAM production was decreased.In vivo evaluation of anti-inflammatory activity as described inExamples can be determined by well-characterized assay. (Gabor, M. MouseEar Inflammation Models and their Pharmacological Appications, 2000).Carrageenan-Induced Paw Edema is a model of inflammation, which causestime-dependent edema formation following carrageenan administration intothe intraplantar surface of a rat paw. The application of arachidonicacid (AA) to the ears of mice produces immediate vasodilatation anderythema, followed by the abrupt development of edema, which is maximalat 40 to 60 min. The onset of edema coincides with the extravasations ofprotein and leukocytes. After one hour the edema wanes rapidly and theinflammatory cells leave the tissue so that at 6 hours the ears havereturned to near normal. These assays, respectively, measure a testcompounds' ability to treat these inflammatory processes via systemicand topical routes of administration.

[0191] MPTP/MPP⁺-induced neurodegeneration of dopaminergic neurons is awell-characterized model that is widely used to understand thepathogenesis of Parkinson's disease. The compounds were tested for theirability to protect against MPTP/MPP⁺ induced neuronal death in vitro andin vivo as shown in the following examples.

[0192] In vitro evaluation of protection against mitochondrialdysfunction is carried out using substantia nigra-derived dopaminergicprogenitor cell line (described in Son J H, Chun et al. J Neurosci, 19:10-20 (1999)) exposed to 1-methyl-4-phenylpyridinium (MPP⁺).

[0193] In vivo evaluation is carried out using mice that have beentreated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), aneurotoxin. MPTP is metabolized into 1-methyl-4-phenylpyridinium (MPP⁺)which then selectively kills dopaminergic neurons thereby inducingParkinsonism. The striking pathologic and clinical similarities betweenidiopathic Parkinson's disease and MPTP-induced Parkinsonism suggestthat the two disorders share common pathogenic mechanism.

[0194] A cellular assay using FRDA-patient derived fibroblasts (asdescribed by Jauslin, ML et al, Human Molecular Genetics 11; 3055-3063(2002)); is used to determine the cell protecting effect of the testcompounds by analyzing survival of skin fibroblasts taken from FRDApatients and unaffected normal donors under conditions of partial GSHdepletion. Exposure of FRDA fibroblasts to BSO (L-buthionine(S,R)-sulfoximine) under conditions of restricted selenium and depletionof cellular glutathione (GSH) causes severe plasma membrane damageleading to cell death. Preincubation with the test compounds before theaddition of BSO is used to determine if they can protect FRDA cells fromBSO-mediated cell death. Cellular GSH content is also directly measuredto exclude the possibility that the compounds may simply preventBSO-mediated GSH depletion.

[0195] Protection against redox stress can be further evaluated in cellculture using either high glutamate induced oxidative stress indopaminergic cell lines or beta-amyloid induced oxidative stress inhippocampal neurons. Using these assays the potency and efficacy of testarticles against redox injury and cell death can be established in ahigh throughput manner as described in the Examples.

[0196] Administration

[0197] The compounds of this invention are administered at atherapeutically effective dosage, e.g., a dosage sufficient to providetreatment for the disease states previously described. Administration ofthe compounds of the invention or the pharmaceutically acceptable saltsthereof can be via any of the accepted modes of administration foragents that serve similar utilities.

[0198] While human dosage levels have yet to be optimized for thecompounds of the invention, generally, a daily dose is from about 0.01to 2.0 mg/kg of body weight, preferably about 0.1 to 1.5 mg/kg of bodyweight, and most preferably about 0.3 to 1.0 mg/kg of body weight. Thus,for administration to a 70 kg person, the dosage range would be about0.7 to 140 mg per day, preferably about 7.0 to 105 mg per day, and mostpreferably about 21 to 70 mg per day. The amount of active compoundadministered will, of course, be dependent on the subject and diseasestate being treated, the severity of the affliction, the manner andschedule of administration and the judgment of the prescribingphysician.

[0199] In employing the compounds of this invention for treatment of theabove conditions, any pharmaceutically acceptable mode of administrationcan be used. The compounds of this invention can be administered eitheralone or in combination with other pharmaceutically acceptableexcipients, including solid, semi-solid, liquid or aerosol dosage forms,such as, for example, tablets, capsules, powders, liquids, suspensions,suppositories, aerosols or the like. The compounds of this invention canalso be administered in sustained or controlled release dosage forms,including depot injections, osmotic pumps, pills, transdermal (includingelectrotransport) patches, and the like, for the prolongedadministration of the compound at a predetermined rate, preferably inunit dosage forms suitable for single administration of precise dosages.The compositions will typically include a conventional pharmaceuticalcarrier or excipient and a compound of this invention or apharmaceutically acceptable salt thereof. In addition, thesecompositions may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, and the like, including, but not limited toanticoagulants, blood clot dissolvers, permeability enhancers and slowrelease formulations.

[0200] Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 90%,preferably about 0.5% to 50%, by weight of a compound or salt of formulaI, the remainder being suitable pharmaceutical excipients, carriers,etc.

[0201] One preferred manner of administration for the conditionsdetailed above is oral, using a convenient daily dosage regimen whichcan be adjusted according to the degree of affliction. For such oraladministration, a pharmaceutically acceptable, non-toxic composition isformed by the incorporation of any of the normally employed excipients,such as, for example, mannitol, lactose, starch, magnesium stearate,sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose,gelatin, sucrose, magnesium carbonate, and the like. Such compositionstake the form of solutions, suspensions, tablets, dispersible tablets,pills, capsules, powders, sustained release formulations and the like.

[0202] Preferably the compositions will take the form of a pill ortablet and thus the composition will contain, along with the activeingredient, a diluent such as lactose, sucrose, dicalcium phosphate, orthe like; a lubricant such as magnesium stearate or the like; and abinder such as starch, gum acacia, polyvinylpyrrolidine, gelatin,cellulose and derivatives thereof, and the like.

[0203] Liquid pharmaceutically administrable compositions can, forexample, be prepared by dissolving, dispersing, etc. an active compoundas defined above and optional pharmaceutical adjuvants in a carrier,such as, for example, water, saline, aqueous dextrose, glycerol,glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, or solubilizingagents, pH buffering agents and the like, for example, sodium acetate,sodium citrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine acetate, triethanolamine oleate, etc. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. Thecomposition or formulation to be administered will, in any event,contain a quantity of the active compound in an amount effective toalleviate the symptoms of the subject being treated.

[0204] Dosage forms or compositions containing active ingredient in therange of 0.005% to 95% with the balance made up from non-toxic carriermay be prepared.

[0205] For oral administration, a pharmaceutically acceptable non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, talcum, cellulosederivatives, sodium crosscarmellose, glucose, sucrose, magnesiumcarbonate, sodium saccharin, talcum and the like. Such compositions takethe form of solutions, suspensions, tablets, capsules, powders,sustained release formulations and the like. Such compositions maycontain 0.01%-95% active ingredient, preferably 0.1-50%.

[0206] For a solid dosage form, the solution or suspension, in forexample propylene carbonate, vegetable oils or triglycerides, ispreferably encapsulated in a gelatin capsule. Such diester solutions,and the preparation and encapsulation thereof, are disclosed in U.S.Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form,the solution, e.g. in a polyethylene glycol, may be diluted with asufficient quantity of a pharmaceutically acceptable liquid carrier,e.g. water, to be easily measured for administration.

[0207] Alternatively, liquid or semi-solid oral formulations may beprepared by dissolving or dispersing the active compound or salt invegetable oils, glycols, triglycerides, propylene glycol esters (e.g.propylene carbonate) and the like, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells.

[0208] Other useful formulations include those set forth in U.S. Pat.Nos. Re. 28,819 and 4,358,603.

[0209] The formulation can be administered in a single unit dosage formfor continuous treatment or in a single unit dosage form ad libitum whenrelief of symptoms is specifically required. For example, theformulation may be administered as a bolus or as a continuousintravenous infusion after onset of symptoms of stroke, myocardialinfarction or chronic heart failure.

[0210] Parenteral administration is generally characterized byinjection, either subcutaneously, intramuscularly or intravenously.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like. In addition, if desired, the pharmaceutical compositions tobe administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,solubility enhancers, and the like, such as for example, sodium acetate,sorbitan monolaurate, triethanolamine oleate, cyclodextrins, etc.

[0211] A more recently devised approach for parenteral administrationemploys the implantation of a slow-release or sustained-release system,such that a constant level of dosage is maintained. See, e.g., U.S. Pat.No. 3,710,795. The percentage of active compound contained in suchparenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject. However, percentages of active ingredient of 0.01% to 10% insolution are employable, and will be higher if the composition is asolid which will be subsequently diluted to the above percentages.Preferably the composition will comprise 0.2-2% of the active agent insolution.

[0212] Nasal solutions of the active compound alone or in combinationwith other pharmaceutically acceptable excipients can also beadministered.

[0213] Formulations of the active compound or a salt may also beadministered to the respiratory tract as an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation have diameters of less than 50 microns,preferably less than 10 microns.

EXAMPLES

[0214] The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

[0215] General Characterization Methods

[0216] Nuclear Magnetic Resonance (NMR) spectra were recorded on aBruker DTX 300 spectrometer using, in most cases, tetramethyl silane(TMS) as the internal reference. Mass spectra were obtained on anAgilent 1100 LC/MSD instrument using either electrospray ionization(positive or negative mode) (ESI) or atmospheric pressure chemicalionization (positive or negative mode) (APCI).

Example 1 Determination of Activity Utilizing-Neuronal Cell Stress Assay

[0217] A. Isolation and Culture of Primary Hippocampal Neuronal Cells.

[0218] Materials

[0219] Neurobasal/B27i: Neurobasal medium (Life Technologies, Rockville,Md.) with 1× B27 supplement (Life Technologies), 0.5 μM L-glutamine, 25μM L-glutamic acid, and 1× Penicillin/Streptomycin.

[0220] Hank's Basic Salt Solution (HBSS, Ca/Mg-free) was prepared bypreparing 1× Hanks CMF (Gibco) supplemented with HEPES (10 mM, pH 7.3),sodium bicarbonate (0.35%), 1× Penicillin/Streptomycin, and 1 mMpyruvate.

[0221] Poly-D-lysine (Sigma, St. Louis, Mo.), 50 μg/ml solution filteredthrough 0.2 μm filter tubes.

[0222] Sigmacote (Sigma, St. Louis, Mo.).

[0223] Plastic Culture Flasks (T75 cm²) or 12-well cell culture platestreated with Poly-D-Lysine (Sigma, St. Louis, Mo.).

[0224] Preparation of Primary Hippocampal Neuronal Cells

[0225] A pregnant female mouse (E18-E19) was euthanized with CO₂ priorto removal of the uterus, which was then placed in a sterile plasticpetri dish. The embryos were removed from the sac, and the embryonicbrains were removed and immersed in cold (4° C.) Buffered Salt Solution(HBSS; Ca/Mg free; Life Technologies) in a small petri dish. Hippocampiwere then removed from the brains under a dissecting microscope and wereplaced on a paraffin-covered dish. The meninges were stripped away andthe dissected hippocampi were collected in a small petri dish in HBSS.The hippocampi were transferred to a 15-ml centrifuge tube (normally10-12 brains) filled with HBSS. The tube containing the brains wascentrifuged at 1000 rpm for 2 min in a tabletop centrifuge. Thesupernatant was removed, 2 ml of HBSS was added to the hippocampi in thetube, and the resulting suspension was triturated 2 times each withlong-tipped siliconized glass pipettes having progressively smallerapertures, starting with a pipette with a standard size opening(approximately 1.0 mm diameter), following with one having an apertureof half standard size (approximately 0.5 mm diameter), then with onehaving an aperture about one-half that size (0.25 mm diameter). Thesuspension was then centrifuged again at 1000 rpm for 2 min in atabletop centrifuge, the supernatant was discarded, and 2 ml ofNeurobasal/B27i (with antibiotics) was added to the tube. Thetrituration procedure described above was then repeated on thissuspension.

[0226] The density of cells was determined on a small aliquot of cellsusing standard counting procedures and correcting for cell viability bytrypan blue stain exclusion. Using this procedure, the expected yield is3×10⁵-6×10⁵ cells/brain. Cells were then added to PDL-coated 24-wellplates, flasks or MetTek dishes in Neurobasal/B27I at a density of about1.5×10⁶ cells (T75 flask) or about 70,000 cells/well of a 24-well plate.Plated cells were incubated at 37 degrees in an atmosphere of 5% CO₂/95%O₂. Media was renewed after 3-4 days by replacing half of it with freshNeurobasal/B27m medium, containing 5 μM cytosine arabinoside (AraC).Seven to eight days from the initial culture, the media was renewedagain, by removing one-half or it and replacing with an equal amount offresh Neurobasal/B27m medium (without Ara-C).

[0227] B. Hippocampal Anoxia-Reoxygenation Cell Death Assay.

[0228] This assay was used to induce ischemia by anoxia-reoxygenation incultured hippocampal neuronal cells. Test compounds were added to assesspotency and efficacy against ischemia-induced neuronal cell injury andcell death.

[0229] Materials.

[0230] Neurobasal media, NoG neurobasal media, B27 supplement and B27Supplement minus AO were obtained from Invitrogen Life Technologies.

[0231] Neurobasal/B27 medium was prepared with 2× B27 minus AOsupplement, 0.5 mM L-glutamine and 0.25× penicillin/streptomycin.

[0232] Cell Tracker Green was obtained from Molecular Probes and a fresh5 μM solution was prepared from 10 mM stock just before use.

[0233] LoG-Neurobasal contains NoG neurobasal medium plus 1 mM glucose,0.5 mM L-glutamine, 0.25× Penicillin/Streptomycin, and 10 mM Hepes (pH7.4).

[0234] Primary hippocampal neuronal cells were prepared according to themethods described above and were cultured in poly-D-lysine coated24-well plates for 10-11 days prior to use.

[0235] Deoxygenated LoG-Neurobasal medium (100 ml) was prepared bypre-equilibrating the medium in a T150 cm² flask in a hypoxic chamberovernight. Following pre-incubation under hypoxic conditions, theLoG-Neurobasal media was lightly bubbled with 100% N₂ for 30 min tocompletely deoxygenate the media. An additional 20 ml LoG-Neurobasal waspre-equilibrated in a T75 cm² flask and was incubated in a normalincubator (5% CO₂) overnight. Reoxygenated medium was prepared byplacing Neurobasa/B27 media overnight in the culture incubator (5%CO₂/95% O₂).

[0236] 10-11 Days after plating the hippocampal neurons, existingculture medium (Neurobasal/B27m) was removed from the cells byaspiration. Cells were washed once with 600 μl/well (24-well cultureplates) of glucose free-BSS. Neurons were replenished with deoxygenatedLoG-Neurobasal (400 μl per well for each well of a 24-well plate). Testcompounds were added directly to each well (usually 3 concentrations ofthe compound plus positive control, each in triplicate). Most testcompounds were dissolved in 100% DMSO; however, concentrations wereadjusted such that the final concentration of DMSO in the cell medianever exceeded 0.5%. Plates containing cells with test compounds wereplaced in a hypoxic chamber for 4-5 hr with plate lids ajar. Fornormoxia controls, pre-equilibrated normoxic LoG-Neurobasal medium wasadded to each well of cells, and the plate was replaced in the normalculture incubator for 4-5 hr. After 4-5 hr of hypoxia, the existingmedia was carefully aspirated off, and 400 μL of new, reoxygenated(pre-equilibrated) Neurobasal/B27 was added to each well. The same testcompounds (in the same the concentrations) were added back into thecorresponding wells. Plates were placed in the cell culture incubator(5% CO₂/95% O₂) and reoxygenated for 20-24 hr. After reoxygenation for20-24 hr, live neurons were quantitated using the cell tracker greenfluorescence method, described below.

[0237] To test for cell viability, existing culture medium was aspiratedfrom each well of the 24 well plates, and neurons were washed once with1 mL of HBSS (pH 7.4, pre-warmed to 30-37° C.). To each well was added500 μL of 5 μM Cell Tracker Green fluorescent dye dissolved in HBSS.Plates were placed in the dark at room temperature for 15 minutes, thenwere washed with 1 mL of HBSS. 500 μL of HBSS was then added to eachwell, and fluorescent cells were counted using a fluorescent microscope.Significantly increased cell viability compared to control cells isindicative of a protective compound.

[0238] Certain compounds of the present invention such as:

[0239] 3-(4-Fluoro-phenyl)-6,7-dimethyl-benzofuran-5-ol;

[0240] 3-(4-Methoxy-phenyl)-6,7-dimethyl-benzofuran-5-ol

[0241] 3-(4-Chloro-phenyl)-6,7-dimethyl-benzofuran-5-ol

[0242] when tested as described above provided protection againststressor-induced cell death in at least about 20% of the cells tested,at concentrations ranging from about 4 to 100 μM.

Example 2 Myocyte Calcium-Contractility Assay.

[0243] A. Isolation and Culture of Primary Neonate Myocytes.

[0244] Materials

[0245] 10× Heart Dissection Solution (HDS) contains the followingcomponents (g/l) in tissue grade water:_NaCl, 68; HEPES, 47.6; NaH₂PO₄,2; Glucose, 10; KCl, 4; MgSO₄, 1, pH adjusted to 7.4. Prior to filtersterilization of diluted (1× HDS) solution, 10 mg phenol red was addedto each 500 milliliters of medium.

[0246] Transferrin and Bovine Insulin were obtained from LifeTechnologies, and resuspended at a concentration of 4 mg/ml in tissueculture grade water.

[0247] DMEM-F12-DMEM/F12, powder, 1:1 containing glutamine andpyridoxine hydrochloride was purchased from Life Technologies. To oneliter equivalent of the powder was added 2.43 g of sodium bicarbonateand 10 ml of 100× Penicillin/Streptomycin in 950 ml of tissue culturegrade water with stirring. The pH was adjusted to 7.2 with 1 M HCl andvolume was adjusted to 1 liter. The solution was filter sterilized then2.5 ml of 4 mg/ml Transferrin, 25011 4 mg/ml Insulin and 30.7 mg ofbromodeoxyuridine were added.

[0248] DMEM-F12 was also prepared 4% FBS for pre-coating the tissueculture plates and initial suspension of the cardiomyocyte pellet.

[0249] Collagenase solution—49 mg of collagenase was resuspended in 120ml 1× HDS.

[0250] Preparation of Primary Neonatal Myocyte Cultures

[0251] Tissue culture ware was pre-coated with DMEM-F12-4% FBS byincubating 50 μl per well of a 96-well plate and 0.25 ml per 12-wellplate at 37° C.

[0252] Two-day old rat pups were removed from their mothers and placedin a sterile container. Pups were dipped quickly into 70% alcohol, thendecapitated and the body was placed in an empty sterile tissue culturedish. An incision was made starting at the neck and progressing towardsthe belly, cutting through the sternum. The heart was removed and placedin a tissue culture dishes containing 1× HDS. The atria were trimmed,and the remaining ventricles were placed into a separate tissue culturedish containing 1× HDS, where they were sectioned into 3-4 pieces each.Ventricles were then transferred to a sterile 250 ml glass flask and the1× HDS was removed. Twenty milliliters of pre-warmed collagenasesolution were added to the ventricles, followed by incubation at 37° C.with shaking. After 30 minutes, the collagenase solution was removed andreplaced with 20 ml fresh pre-warmed collagenase. Incubation wascontinued for an additional 30 minutes. At the end of the incubation,any tissue chunks were allowed to settle prior to removing thecollagenase (containing the isolated cardiomyocytes) from the disruptedtissue pieces. The isolated myocytes were added to a 50 ml Falcon tubecontaining 2 ml Fetal Bovine Serum (FBS). The remaining tissue pieceswere subjected to a second digestion by adding 20 ml fresh pre-warmedcollagenase and incubating as above for 30 minutes. This second digestwas then centrifuged at 1000 rpm for 10 minutes (tabletop centrifuge).The resulting supernatant was discarded, and the cell pellet wassuspended with 4 ml FBS. The resulting cell suspension was placed in theincubator at 37° C. This step was repeated several additional times toharvest additional material.

[0253] Percoll gradients were prepared by adding 2.5 ml of 10× HDS to22.5 ml of Percoll (Life Technologies) with mixing (Percoll Stock). TopGradient solution (11 ml Percoll Stock and 14 ml 1× HDS) and BottomGradient solution (13 ml Percoll Stock and 7 ml 1× HDS) were prepared.Four milliliters of the Top Gradient solution were transferred into 6×15ml sterile Falcon tubes. Three milliliters of the Bottom Gradientsolution were placed in each tube by inserting a serological pipette tothe bottom of the tube and slowly adding the liquid.

[0254] All the digests (5) were pooled in one 50 ml Falcon tube andcentrifuged on a tabletop centrifuge at 1000 rpm for 10 minutes. Thesupernatant was discarded, and the cell pellet was resuspended in 12 mlof 1× HDS. Two milliliters of the cell suspension was added to the topof each gradient. The gradient tubes were then centrifuged at 3000 rpmfor 30 minutes without braking in a Beckman Allegra 6 centrifuge (GH3.8A rotor). Following centrifugation, the cells segregated into twosharp bands at the two interfaces. The lower band of the two bands wasenriched for cardiomyocytes; there was also a cardiomyocyte pellet atthe bottom of the tube. The upper band was enriched for fibroblasts andother non-cardiomyocytes. The upper portion of the gradient wasaspirated down to just above the cardiomyocyte layer. The cardiomyocytelayer was then carefully removed along with the pellet, and the twofractions were pooled in a sterile 50 ml Falcon tube, along withcorresponding fractions from additional gradient tube; then 1× HDS wasadded to a total volume of about 50 ml. The tube was centrifuged at 1000rpm for 7 minutes. The supernatant was discarded and resuspended in 25ml 1× HDS. A further 25 ml of 1× HDS was added and the centrifugationstep was repeated. The cell pellet was resuspended carefully butthoroughly in 40-50 of DMEMF12-4% FBS.

[0255] A small aliquot of the cell suspension was counted in ahemocytometer. The DMEM/F12-FBS coating medium was aspirated from thetissue culture dishes. The cardiomyocytes were added to the dishes at aplating density of 7.5×10⁴/well per 96-well in 200 μL and 1.5×10⁵/wellper 12-well in 3 ml. The cultures were incubated at 37° C. with 5% CO₂overnight. The original medium was removed, and add fresh DMEM/F12-5%FBS was added to each culture, prior to incubation at 37° C. with 5% CO₂for a further 48 hours, before use.

[0256] B. Contractility Assay

[0257] Materials

[0258] Complete DMEM-F12: DMEM/F12, powder, 1:1 containing glutamine andpyridoxine hydrochloride was purchased from Life Technologies(Invitrogen Life Technologies, Carlsbad, Calif.). Powder sufficient toprepare one liter of buffer and 2.43 g of sodium bicarbonate was mixedinto 950 ml of tissue culture grade water. The pH was adjusted to 7.2with 1 M HCl and the remaining water was added to make 1 liter.Following filter sterilization, 10 ml of 100× Penicillin/Streptomycin,2.5 ml of 4 mg/ml Transferrin, 250 μl 4 mg/ml Insulin and 30.7 mg ofbromodeoxyuridine were added, and the mixture was incubated at 37° C.prior to use.

[0259] 1 mM glucose in DMEM was made from DMEM without L-glutamine,without glucose, without sodium pyruvate, purchased from LifeTechnologies.

[0260] 20 μM Fluo-4: Cell permanent AM ester of Fluo-4 was obtained fromMolecular Probes (Eugene, Oreg.) as a dry powder to be stored at −20° C.This fluorescent dye is light sensitive and should be made up fresh at 1mM in DMSO prior to use to prevent light degradation.

[0261] 10 mM CaCl₂ solution was made fresh each day in 1× HBSS andincubated at 37° C. prior to use.

[0262] Neonatal cardiomyocytes were isolated as described above. Thecardiomyocytes were plated in 96-well format (black clear-bottomedplates) at a density of 7.5×10⁴ per well and grown for 2 days in thepresence of 5% FBS prior to use in the assay.

[0263] Physiological ischemia was simulated by placing thecardiomyocytes in an anaerobic chamber (0% O₂, 85% N₂, 5% CO₂ & 10% H₂)in DMEM containing 1 mM glucose. Positive control cells are treated withDMEM-F12 containing 25 mM Glucose, which protects against the anoxia.

[0264] The test compounds were made up in DMEM-1 mM glucose in 96deep-well mother plates and appropriately diluted for use in the assay.The media was removed from the cells and replaced with 200 μl of eitherDMEM-F12 or 1 mM DMEM with or without test compounds. The plates werethen placed inside the 37° C. incubator in the anaerobic chamber andincubated for 16 hours. The plates were then removed and reoxygenated bythe addition of DMEM-F12. The DMEM with or without test compounds iscarefully removed from the cells and replaced with pre-warmed DMEM-F12containing 5% FBS. Since the anoxic treatment may damage and/or kill thecells, causing them to dislodge from the bottom of the wells gentleaspiration of media is required at this step. The cells were then placedin a normal incubator at 37° C. and incubated for two hours to allow thecells to reoxygenate.

[0265] A working solution of 20 μM Fluo-4 was added to pre-warmed 1×HBSS. The cells were loaded with Fluo-4 by first removing media from thecells and replacing with 100 μl of 20 μM Fluo-4. Unloaded control cellswere treated in parallel with 1× HBSS alone. All cells were thenincubated at 37° C. for 30 minutes. Before fluorescence measurementswere made, the cells were washed in indicator-free medium (HBSS) toremove any dye that is non-specifically associated with the cellsurface. Cells were then incubated for an additional 20 minutes at roomtemperature. Basal Fluo-4 fluorescence was measured using the 485 nmexcitation and 538 nm emission filter pair on a microplate flourometer(Fluorskan™, Thermo Labsystems Oy, Helsinki, Finland). Each well wasread for 160 ms to obtain a baseline reading, then stimulated tocontract by addition of 10 mM CaCl₂. Following incubation at 37° C. for30 minutes, a stimulated fluorescence was taken after 90 minutes.

[0266] Compounds of the present invention such as

[0267] (5-Hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone;

[0268] Acetic acid6,7-dimethyl-2-(2-morpholin-1-yl-acetyl)-benzofuran-5-yl ester;

[0269]2-(1-Hydroxy-2-morpholin-4-yl-ethyl)-6,7-dimethyl-benzofuran-5-ol;

[0270] Acetic acid 2-(2-bromo-acetyl)-6,7-dimethyl-benzofuran-5-ylester;

[0271]1-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-2-morpholin-4-yl-ethanone;

[0272] 1-(4-Bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone;

[0273] 1-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone;

[0274] 4-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-benzonitrile;

[0275] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde;

[0276] 2-Hydroxymethyl-6,7-dimethyl-benzofuran-5-ol;

[0277] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester;

[0278] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid;

[0279] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acidbis-(2-hydroxy-ethyl)-amide;

[0280] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester; and

[0281] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acidbis-(2-hydroxy-ethyl)-amide;

[0282] when tested as described above show a protection of at least 20%and the presence of calcium transients in amounts indicative of abilityguard against ischemic damage and allow the cells to maintain theircontractile function.

Example 3 Rat Middle Cerebral Artery Occlusion (MCAO) Model of CerebralIschemia

[0283] A. Animal Preparation

[0284] Male Wistar rats (Harlan, Ind.) weighing 300-350 g are commonlyused in these experiments. Animals are allowed free access to water andcommercial rodent diet under standard laboratory conditions. Roomtemperature is maintained at 20-23° C. and room illumination is on a12/12-hour light/dark cycle. Animals are acclimatized to the laboratoryenvironment 5 to 7 days prior to the study, and fasted (with free accessto water) overnight before surgery.

[0285] B. Middle Cerebral Artery Occlusion (MCAO)

[0286] Anesthesia is maintained by inhalation of 3.0% isoflurane(Aerrane, Front Dodge, Iowa) in 0.8% oxygen. The animal's neck wasshaved and sterilized before operation. Body temperatures werecontrolled and maintained at 37.5° C. +/−1 degree via external heatingand cooling devices. To lower the body temperature, animals are placedin a cooling chamber, which uses ice to cool circulating air. Throughoutthe study the body temperature is recorded using a temperaturetransponder (BMDS Inc., Seaford, Del.) implanted subcutaneously at thetime of MCAO between the rat shoulder blades that allows the user toread the body temperature via a pocket scanner (BMDS Inc., Seaford,Del.). The body temperature may also be taken by inserting thetemperature probe into the animal's rectum. Body temperature is recordedevery hour for 6 hours post-occlusion; however, body temperatures weretaken more frequently so that they could be maintained at thenormothermic temperature.

[0287] Animals were subjected to two hours MCAO using a modifiedintraluminal filament technique, as follows: A midline incision on theventral part of the neck is made to expose external and internal carotidarteries. The right external and common carotid arteries are ligated bya suture (silk 5/0, Carlisle Laboratories, Farmers Branch, Tex.) and theright internal artery is temporarily ligated using a microvascular clip(Fine Science Tool Inc., Foster City, Calif.). A small incision was madein the common carotid artery. A nylon filament, its tip rounded byheating, is prepared from a fishing line (Stren Fishing Lines,Wilmington, Del.) and is inserted from the right common carotid artery.The filament is advanced into the internal carotid artery 18-20 mm fromthe point of bifurcation of internal and external arteries and a sutureis tightly ligated around the filament. Two hours post occlusion,animals are re-anesthetized to allow reperfusion for the remaining ofthe experiment by removal of the filament.

[0288] C. Drug Administration

[0289] Test compounds may be administered by any of a number of routes,such as those described below. Compounds can be administered before,during or after occlusion, as appropriate to the protocol.

[0290] a) Intracerebroventricular (ICV) Infusion

[0291] The anesthetized animal is placed on a stereotaxic apparatus(Harvard Apparatus, S. Natick, Mass.). Anesthesia is maintained byinhalation of 3.0% isoflurane (Aerrane, Front Dodge, Iowa) in 0.8%oxygen throughout the entire procedure. The scalp is shaved andsterilized prior to surgery. A midline sagittal incision about 3 cm longis made slightly behind the eyes to expose the skull. The skull isscraped with a rounded end spatula to remove periosteal connectivetissue. A bur hole is placed 1.5 mm lateral, 1 mm posterior to the leftof the bregma to mark the left lateral ventricle. A brain infusioncannula (ALZET Co., Palo Alto, Calif.) is inserted 4 mm deep into thehole. The desired depth is adjusted by attaching spacers to the cannula.The cannula attached to a 4-cm silastic catheter (Helix Medical Inc.,Carpinteria, Calif.) fixed in place with dental cement (Ketac-cement,Norristown, Pa.). The catheter is either attached to a primed osmoticpump placed subcutaneously between the shoulder blades for permanentinfusion or to a syringe for a short infusion.

[0292] b) Intravenous (IV) Osmotic Pump Implantation into the JugularVein

[0293] Anesthesia is maintained by inhalation of 3.0% isoflurane(Aerrane, Front Dodge, Iowa) in 0.8% oxygen throughout the entireprocedure. The animal's neck will be shaved and sterilized beforeoperation. A midline incision is made on the ventral part of the neck toexposes the jugular vein. The vein is isolated and ligated with a suture(silk 5/0, Carlisle Laboratories, Farmers Branch, Tex.) rostral to thepoint of the incision and a microvascular clip (Fine Science Tool Inc.,Foster City, Calif.) close to the heart. A small incision is madebetween two ligations. A 2-cm silastic catheter (Helix Medical Inc.)attached to a PE-60 tube (Becton. Dickinson and Co. Sparks, Md.)connected to an ALZET (ALZET CO. Palo Alto, Calif.) pump is introducedand advanced 2 mm into the jugular vein toward the heart. Themicrovascular clip is removed and the catheter is secured in place witha suture (silk 5/0, Carlisle Laboratories, Farmers Branch, Tex.). Thepump is placed into a pocket made subcutaneously between the shoulderblades, allowing the catheter to reach over neck to the jugular veinwith sufficient slack to permit free movement of neck and head.

[0294] c) IV Infusion Via Femoral Vein

[0295] Anesthesia is maintained by inhalation of 3.0% isoflurane(Aerrane, Front Dodge, Iowa) in 0.8% oxygen throughout the entireprocedure. The exterior site of the right femoral vein is shaved andsterilized prior to surgery. A 3-cm incision is made in the right groinregion and the femoral vein is isolated. A small incision is made on thefemoral vein temporarily ligated with a microvascular clip to introduceand advance a polyethylene (PE-50) catheter (Becton Dickinson and Co.Sparks, Md.). The catheter is secured in place with suture (silk 5/0,Carlisle Laboratories, Farmers Branch, Tex.). The other end of thecatheter is attached to a syringe filled with the heparinized saline forthe bolus injection. Using a hemostat, a pocket is made subcutaneouslyon the back of the animal so the PE catheter can be brought up to theexteriorization point at the nape of the neck for either a bolusinjection or a continuous injection by an osmotic pump.

[0296] d) Intraperitoneal (IP) Injection

[0297] An awake rat is held in a standard hand hold position, a 23 3/4 Gneedle is injected into the lower right quarter of the abdomen pass theperitoneum, slightly off the midline. To avoid organ injection, theplunger of the syringe is slightly pulled back. If no fluid iswithdrawn, the content of the syringe is delivered into the abdominalcavity.

[0298] D. Behavioral Assessment

[0299] One hour after MCAO, the animal was gently held by its tail andobserved for forelimb flexion. Then the animal is placed on the floor tobe observed for walking pattern; only the animals that score 3 onBederson grading system (Table 1) are included in the study. TABLE 1Bederson Grading System for Neurological Evaluation Neurological deficitGrading Behavioral observation Normal grade 0: No observable deficitModerate grade 1: forelimb flexion Severe grade 2: forelimb flexion,decreased resistance to lateral push Extreme grade 3: forelimb flexion,decreased resistance to lateral push, circle to paretic side

[0300] E. Evaluation of Ischemic Damage

[0301] Twenty-four hours post-MCAO, or longer, in some experiments,animals were sacrificed by CO₂ asphyxiation (dry ice). The brain wasquickly removed from the skull, using standard procedures, rinsed inchilled saline solution, and placed on a rat brain tissue slicer (ASIinstrument, MI). Seven 2-mm thick coronal slices are cut from each brainusing razor blades. The slices were immersed in 0.9% saline containing1.0% 2,3,5-triphenyltetrazolume chloride (TTC) (Sigma Chemical Co., St.Louis, Mo.) and incubated in a 37° C. water bath for 30 minutes.

[0302] After staining, each 2-mm slice is photographed with a TMC-7camera (JH Technologies, Ca) which is directly connected to a desktop PCto capture and save the image of each brain slice. This image is usedfor the measurements of the regions of interest using a computer-basedimage processing system (Metamorph).

[0303] To measure each area, the region of interest is selected using afreehand selection tool, the area is automatically computed by selectingthe measure command. The measurements for primary regions of interestare right hemisphere, left hemisphere, total infarct, subcorticalinfarct, total penumbra and subcortical penumbra. After all regions ofinterest are measured for all seven slices of the brain, they are sortedby slice number and the corresponding regions of interest using anExcell macro called statistic final. This macro also calculates thecortical penumbra, cortical infarct and total ischemic damage for eachslice; the corresponding areas of each rat brain will be added togetherto produce a single measurement for each area. Since the ipsilateralhemisphere is swollen following MCAO, edema volume is calculated andreported as the volumetric differences between the right and lefthemispheres of each brain slice. Using the % of hemispheric swelling allthe volumes will be corrected for the edema. The volume of the damage isdetermined using the calculations below for each rat's brain.Measurement Equation Corrected Value(s) Cortical Penumbra (C.P.) TotalPenumbra-Subcortical Total Penumbra (T.P._(corr)) = (T.P. × Penumbra %H.S./100) C.P._(corr.) = C.P. − (C.P. × % H.S./100) S.P._(corr.) = S.P.− (S.P. × % H.S./100) Cortical Infarct Total Infarct − SubcorticalInfarct T.I._(corr.) = T.I. − (T.I. × % H.S./100) S.I._(corr.) = S.I. −(S.I. × % H.S./100) C.I._(corr.) = C.I. − (C.I. × % H.S./100) TotalIschemic Damage (T.I.D.) Total Penumbra + Total Infarct T.I.D._(corr.) =T.I.D. − (T.I.D. × % H.S./100) Total Volume (mm³) Each value ismultiplied by 2 (the thickness of the tissue). Edema Volume Thevolumetric differences between the sum of right and left hemispheresdetermines the edema volume. % Hemispheric swelling (H.S.) Edema ×100/left hemisphere

[0304] F. Statistical Analysis

[0305] Sample size is chosen to achieve a 90% probability of significantresults. The measurements, which represented the same region of interestin seven slices of each rat's brain are added together to yield a singlemeasurement for total infarct, subcortical infarct, cortical infarct,total penumbra, subcortical penumbra, cortical penumbra, total ischemicdamage and edema in each animal. Group data are presented as means +/−SEM. Differences at the level of p<0.05 are considered statisticallysignificant. Between groups comparison of each region of interest arecarried out by unpaired student t test (between two groups) or one wayANOVA followed by post hoc Bonferroni's multiple comparisons or by thenonparametric Dunnett's test (between control and the drug treatedgroups).

[0306] Compounds of the present invention can be tested as describedabove.

Example 4 FRDA Fibroblast Assay for Protection from Oxidative Stress

[0307] A. Cell Culture and Reagents

[0308] Primary fibroblasts were derived from donors with a moleculardiagnosis of FRDA and control donors with no mitochondrial disease. Celllines C1 and F1 were provided by the Swiss Network on Friedreich AtaxiaResearch, line F3 was provided by Hopital Necker, Paris (France), andlines F2, C2 and C3 were obtained from Coriell Cell Repositories(Camden, N.J., USA; catalog nos GM04078, GM 08402 and GM08399,respectively). All cell types were diagnosed at the molecular level forintronic GAA triplet repeat length using a PCR-based method, accordingto methods known in the art. FRDA-cell types had ˜400-450 repeats (F2line) or more (F1 and F3), whereas control cell lines displayed PCRproducts of normal length. The cells were seeded in microtiter plates ata density of 4000 cells per 100 μl in growth medium consisting of 25%(v/v) M199 EBS and 64% (v/v) MEM EBS without phenol red (Bioconcept,Allschwil, Switzerland) supplemented with 10% (v/v) fetal calf serum(PAA Laboratories, Linz, Austria), 100 U/ml penicillin, 100 μg/mlstreptomycin (PAA Laboratories, Linz, Austria), 10 μg/ml insulin (Sigma,Buchs, Switzerland), 10 ng/ml EGF (Sigma, Buchs, Switzerland), 10 ng/mlbFGF (PreproTech, Rocky Hill, N.J., USA) and 2 mM glutamine (Sigma,Buchs, Switzerland). The cells were incubated in the presence of thevarious test compounds for 24 h before addition of BSO (L-buthionine(S,R)-sulfoximine).

[0309] B. Cell Viability Measurements

[0310] Cell viability was measured after the first signs of toxicityappeared in the BSO-treated controls (typically after 16-48 h). Thecells were stained for 60 min at room temperature in PBS with 1.2 μmcalceinAM and 4 μm ethidium homodimer (Live/Dead assay, MolecularProbes, Eugene, Oreg., USA). Fluorescence intensity was measured with aGemini Spectramax XS spectrofluorimeter (Molecular Devices, Sunnyvale,Calif., USA) using excitation and emission wavelengths of 485 and 525nm, respectively. Live cell imaging was performed with a Zeiss Axiovert135 M fluorescence microscope equipped with a cooled CCD camera(Sensicam, PCO Computer Optics, Kelheim, Germany). Image acquisition wasperformed with the ImagePro Plus software (Media Cybermetics, SilverSpring, Md., USA).

[0311] C. Glutathione Content

[0312] Cellular GSH content was measured to exclude the possibility thatthe compounds simply prevented BSO-mediated GSH depletion. Cells wereremoved from 100 mm culture dishes by trypsinization, washed twice withPBS, snap-frozen in 100 μl PBS and lysed in PBS supplemented with aprotease inhibitor cocktail (Complete, Roche Diagnostics, Rotkreuz,Switzerland) by four freeze-thaw cycles. Total protein content wasmeasured with the BioRad protein assay (BioRad, Hercules, Calif., USA).Reduced glutathione content was determined essentially as described byKamencic et al. (2000) with a final monochlorobimane (mCIB, MolecularProbes, Eugene, Oreg., USA) concentration of 25 μM. The GSH-mCIB adductfluorescence was measured with a Gemini spectrofluorimeter usingexcitation and emission wavelengths of 380 and 470 nm, respectively.

[0313] D. Glutathione Peroxidase Assay

[0314] Cell extracts obtained for total GSH measurements were adjustedto a final protein concentration of 1.85 mg/ml. Enzymatic activity wasmeasured with the Glutathione peroxidase cellular activity assay kit(Sigma, St Louis, Mo., USA) according to manufacturer's instructionswith minor modifications. Enzymatic activity was measured with 40 μgtotal protein extract in a final reaction volume of 100 μl monitoringNADPH consumption as a decrease in NADPH fluorescence. GPX enzymaticactivities were determined by measuring the maximum velocity of theNADPH consumption using tert-butyl hydroperoxide as substrate andpurified bovine erythrocytes GPX (Sigma, St Louis, Mo., USA) asstandard. All measurements were done in triplicate in 96-well plateswith a Gemini spectrofluorimeter using excitation and emissionwavelengths of 340 and 445 nm, respectively.

[0315] E. Data and Statistics

[0316] In experiments carried out in support of the present invention,certain compounds such as

[0317] 5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester;

[0318] 5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid3,7-dimethyl-octa-2,6-dienyl ester;

[0319] 2-Hydroxymethyl-6,7-dimethyl-benzofuran-5-ol;

[0320] (5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone; and

[0321] 3-amino-5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acidmethyl ester significantly reduced cell death in FRDA fibroblastscompared to untreated FRDA fibroblasts with an EC₅₀ of between about 0.8μM and 27 μM.

Example 5 Evaluations of Sensorimotor Behavior

[0322] A. Fore and Hindlimb Grip Strength Test in Rats

[0323] Animals with cerebral infarction induced by transient orpermanent unilateral occlusion of the middle cerebral artery (MCA) andsham-operated rats are tested for grip strength, a standard model ofneuromuscular function and sensorimotor integration, using aComputerized Grip Strength Meter for Rats (Dual Stand Model, ColumbusInstruments, Columbus, Ohio).

[0324] Animals are moved into the testing room for 30 minutes beforetesting. Prior to testing, each gauge is calibrated with a set of knownweights and the apparatus is adjusted for the size of animal, accordingto manufacturer's instructions. The forelimb measurements are carriedout with the meter in the tension peak mode to freeze the reading as thesubject is pulled away from the grip bar. The hindlimb measurements arecarried out with the meter in the compression peak mode to freeze thereading as the subject's hindlimbs are pulled over the bar toward themeter. Each animal is hand-held by the investigator while pulled pastthe grip bars, using a consistent technique, leaving the fore and hindlimbs free to grasp the grip bars.

[0325] Testing is carried out on postoperative day 2 and repeated, in ablind-randomized fashion, twice weekly for a defined interval.Typically, three successive readings are taken for each animal with anintertrial interval long enough to record the data and zero both metersfor the next trail.

[0326] B. Rota-Rod Test in Rats

[0327] Apparatus: Rota-Rod Treadmill for Rats (7750 Accelerating Model,from UGO BASILE, COMERIO-ITALY).

[0328] Procedure: Animals with cerebral infarction induced by transientor permanent unilateral occlusion of the middle cerebral artery (MCA)and sham-operated rats are tested in this study, using a Rota-RodTreadmill for Rats (7750 Accelerating Model, UGO Basile, Comerio,Italy). The animals are moved into the testing room 30 minutes beforetesting. Every rat receives 2-3 training runs of 1-2 minutes atintervals of 2-3 hours before testing. The cylinder on the apparatus isset in motion before placing the rats in position. The motor is set at aconstant selected speed in 7700 on RESET mode, and the rats are placed,one by one, in their sections.

[0329] Testing is carried out on postoperative day 2 and repeated, in ablind-randomized fashion, twice weekly for a defined interval.Typically, three successive readings are taken for each animal with anintertrial interval long enough to record the data and zero both metersfor the next trail.

Example 6 High Glutamate-Induced Oxidative Stress Assay

[0330] This procedure was used to induce high glutamate oxidative stress(HGOS) in a dopaminergic neuronal cell line. Using this assay thepotency and efficacy of test articles against HGOS neuronal cell injuryand cell death was established in a high throughput manner.

[0331] Materials

[0332] Dopaminergic neuronal cell line

[0333] DMEM-No Glucose (Life Technologies Cat #11966-025)

[0334] L-glutamine (Life Technologies Cat #25030-081)

[0335] L-glutamic acid, monosodium salt (Sigma Cat #G5889)

[0336] D-glucose (Sigma Cat #G-6151)

[0337] 10× HBSS buffer(pH 7.4) (950 ml Pyrogen-free water, 2.44 g/LMgCl2.6H2O, 3.73 g/L KCl, 59.58 g/L Hepes, 58.44 g/L NaCl, 1.36 g/LKH2PO4, 1.91 g/L CaCl2.2H2O and pH to 4.5 with HCl)

[0338] Cell Tracker Green fluorescent dye (Molecular Probes, Cat #2925).Prepare a 5 μM solution in pre-warmed HBSS just prior to use.

[0339] Sterile 96-well plates precoated with poly-D-lysine (CorningCatalog #3665)

[0340] 96-well deep well mother plate, DyNA Block 1000 (VWR Catalog#40002-008)

[0341] Neuronal Cells

[0342] The cells were seeded into 96-well plates at a density of 2000per well and left to grow for 72 hours in a 33° C. incubator with 5% CO₂in air atmosphere. The passage number of the cells for each assayexperiment were no later than p11 in order to minimize experimentalvariation.

[0343] Compound Preparation in Deep-Well Mother Plates

[0344] VWRBrand DyNA Block 1000, deep well mother plates (VWR Cat.#40002-008) were used for the preparation of the test compounds.

[0345] All compounds were dissolved in DMEM-No Glu containing 1 mMglucose, 30 mM glutamate and 1× Pen/Strep. DMEM-No Glu with 1 mM glucoseand 1× P/S was used as the negative control, DMEM-No Glucose with 1 mMglucose, 100 M glutamate was used as a positive control and 100 μMGlutathione was added to the positive control as a standard. All of theprocedures for this involving the making and dilution of compounds wereperformed using aseptic conditions and with minimal light.

[0346] Cell Preparation

[0347] The plates were removed from the incubator and examined under themicroscope for morphological appearance and density. Using an aseptictechnique and an 8-channel aspirator the media was carefully removedfrom the cells and replaced with 200 μl of 1× HBSS. This was done asquickly as possible to prevent the cells drying out. The plates werethen placed in the humidified 37° C. incubators of the Biomek 2000 SideLoader. Four plates were washed at a time so as to minimize the timethat the cells were sitting in 1× HBSS prior to addition of the compoundtest solution.

[0348] Experimental Setup

[0349] The Beckman Biomek workstations were used to load the compoundsand controls from the mother plates onto the cell plates that wereprewashed with HBSS under sterile conditions. The plates were incubatedin the upper HTS incubator at 37° C. in 5% CO₂ for exactly 16 hrs. Thefollowing day, using the Beckman Biomek workstations, the plates wereremoved from the incubator. Using Cell Tracker Addition, the compoundswere removed from the plates, washed once with 200 μM of pre-warmed 1×HBSS and then 100 μL of 5 μM Cell Tracker Green was added to each well.The plates were incubated at 37° C. for 30 min to allow the dye to enterthe cell and be cleaved by the esterases. After washing the cells twicewith prewarmed 1× HBSS, the plates were read with the 485 excitation;538 emission filter pair on a Fluoroskan.

[0350] Certain compounds of the present invention such as:

[0351] 6,7-dimethyl-2-(4-nitro-phenyl)-benzofuran-5-ol;

[0352] 4-(5-H\hydroxy-6,7-dimethyl-benzofuran-2-yl)-benzonitrile;

[0353] 6,7-dimethyl-3-phenyl-benzofuran-5-ol;

[0354] 2-hydroxymethyl-6,7-dimethyl-benzofuran-5-ol;

[0355] 2-bromo-1-(5-acetoxy-6,7-dimethyl-benzofuran-2-yl)-ethanone; and

[0356] (5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone;

[0357] were considered to be active when they exhibited protectionagainst HGOS cell injury and cell death at an EC₅₀ of less than 30 μM,preferably less than 10 μM.

Example 7 MPP⁺ Cell Death Assay

[0358] Media Composition

[0359] RF media: DMEM-No glucose, glucose (29.1 mM), L-glutamine (1.4mM), 10% heat-inactivated FBS, and 1× penicillin/streptomycin (P/S)

[0360] Wash media: DMEM-No glucose and 1× P/S

[0361] Low serum media: DMEM-No glucose, glucose (29.1 mM), L-glutamine(1.4 mM), 0.5% FBS, and 1× P/S

[0362] Assay Media: DMEM-No glucose, L-glutamine (1.4 mM), 0.5% FBS, and1× P/S

[0363] Experimental Procedure

[0364] The substantia nigra-derived dopaminergic progenitor cell linewas seeded in poly-D-lysine-coated 24-well plates at a density of 4500cells per well in RF media. The cells were left to attach for 16 hoursin a 33° C. incubator (5% CO₂) after which time they were washed oncewith 500 μL wash media and then differentiated into a neuronal phenotypeby incubating in low serum media for 24 hours in a 39° C. incubator (5%CO₂).

[0365] After 24 hours the low serum medium was aspirated from the cellsand the monolayer was washed once with 500 μL wash media. Test articleswere diluted to 2-fold the desired testing concentration in assay mediaand 250 μL was added to the cells. From a 10 mM stock, a workingsolution of 140 μM 1-methyl-4-phenylpyridinium (MPP⁺) (Sigma, St. Louis,Mo.) was made in assay media and 250 μL of this working solution wasalso added to the cells. The final volume in each well was 500 μL andthe final concentration of MPP⁺ was 70 μM. As a negative control, cellswere incubated with 500 μL assay media with no additions.

[0366] Cells were incubated in a 39° C. incubator (5% CO₂) for 24 hours.After this time, the number of live neurons remaining in each well wasdetermined using a fluorescent vital cell stain, Cell Tracker Green(Molecular Probes, Eugene, Oreg.). Assay media was aspirated from thecells and 400 μL of 2.5 μM Cell Tracker Green was added to each well.Cells were placed in a 37° C. incubator for 5 minutes after which timethe cell stain was aspirated off and 500 μL of HBSS (Invitrogen LifeTechnologies, Carlsbad, Calif.) was added to each well. The number oflive cells in each well was then quantitated using an automatedfluorescent microscope/imaging system (Universal Imaging, DowningtownPa.).

[0367] Results:

[0368] Certain compounds of the present invention when tested asdescribed above provided protection in at least 30% of the cells testedat concentrations ranging from about 0.1 to 10 μM.5-Hydroxy-3,6,7-trimethyl-benzofuran-2-yl)phenyl-methanone providedprotection in 78% of the cells at a concentration of 2 μM, and1-(3-Bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone provided in65% of the cells at a concentration of 100 μM.

Example 8 Beta-Amyloid Cell Death Assay

[0369] Media Composition

[0370] Neurobasal/B27i: Neurobasal medium plus 1× B27 supplement, 0.5 mML-glutamine, 25 μM L-glutamic acid, and 0.5× Penicillin/Streptomycin

[0371] Neurobasal/B27m: Neurobasal medium plus 1× B27 supplement and 0.5mM L-glutamine

[0372] BSS (Ca/Mg free): HBSS (calcium/magnesium free) plus 10 mM Hepes(pH 7.25), 1× Penicillin/Streptomycin, and 1 mM Sodium Pyruvate

[0373] Glucose-free BSS_(O): 143.6 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂,0.8 mM MgSO₄, 1 mM NaH₂PO₄, 26.2 mM NaHCO₃, 10 mg/l phenol red, 0.25×Penicillin/Streptomycin, and 10 mM Hepes (pH 7.4)

[0374] Papain Quench solution: Neurobasal medium plus 1× B27 supplement,1× Penicillin/Streptomycin and 0.5 mg/ml DNase1

[0375] Assay media: Neurobasal medium plus 1× B27 (minus AO) supplement,0.5 mM L-glutamine, and 0.25× Penicillin/Streptomycin.

[0376] Experimental Procedure

[0377] Hippocampal Cell Culture

[0378] Hippocampal neurons were isolated from E18 rat embryos asfollows. Embryos were decapitated and the heads immersed in cold BSS(Ca/Mg free). Using a dissecting microscope the hippocampi weredissected out and placed in cold BSS (Ca/Mg free). The isolatedhippocampi were then centrifuged at 1000 rpm for 2 min, the BSSaspirated off and 2 ml of 2 mg/ml Papain in Neurobasal media added per10 embryos. After mixing on a rotational shaker for 10 min at 37° C., 5ml Papain Quench solution was added. Cells were then centrifuged at 1000rpm for 2 min, the supernatant was aspirated and 2 ml of Neurobasal/B27iwas added. The cells were triturated 6 times with siliconized pipettes(decreasing bore size) after which an additional 5 ml Neurobasal/B27iwas added. The cell suspension was then centrifuged at 1000 rpm for 2min, the supernatant was aspirated and 2 ml of Neurobasal/B27i wasadded. Cells were titurated again as described above and the volume ofNeurobasal/B27i was adjusted to 1 ml/embryo. Cells were then counted andseeded at a density of 75,000 cells per well in a poly-D-lysine coated24-well plate. After four days media was removed from the cells andreplaced with Neurobasal/B27m media +5 μM Ara-C (cytosine arabinoside).Seven days after isolation the media was removed again and replaced withfresh Neurobasal/B27 m media. Ten days after isolation the hippocampalcultures were used in the assay described below.

[0379] Preparation of oligomeric beta-amyloid (AO) peptide

[0380] Aggregation of Aβ(1-42) (American Peptide) into oligomers wascarried out according to the method of Dahlgren et al, (2002) J. BiolChem 277: 32046-32053. The Aβ peptide was dissolved to 1 mM inhexafluoroisopropanol (HFIP) and aliquoted into sterile microcentrifugetubes. The HFIP was removed under vacuum and the peptide film stored at−20° C. The day before the assay, the peptide film was resuspended indry DMSO to a concentration of 5 mM. Ham's F-12 media was then added tobring the peptide to a final concentration of 100 μM, and this solutionwas incubated at 4° C. for 24 hours to allow formation of oligomers.

[0381] Treatment of Hippocampal Neurons with Oligomeric Aβ

[0382] The existing growth medium was aspirated from the hippocampalcultures and the monolayer was washed once with 500 μl glucosefree-BSS₀. Test articles were diluted to 2-fold the desired testingconcentration in assay media and 250 μL was added to the cells. From the100 μM oligomeric beta-amyloid peptide solution described above, aworking solution of 6 μM was made in assay media. 250 μL of this workingsolution was also added to the cells. The final volume for each well was500 μL and the final concentration of Aβ peptide was 3 μM. As a negativecontrol, cells were incubated with 500 μL assay media with no additions.

[0383] Cells were incubated in a 39° C. incubator (5% CO₂) for 24 hours.After this time, the number of live neurons remaining in each well wasdetermined using a fluorescent vital cell stain, Cell Tracker Green(Molecular Probes, Eugene, Oreg.). Assay media was aspirated from thecells and 400 μL of 2.5 μM Cell Tracker Green was added to each well.Cells were placed in a 37° C. incubator for 5 minutes after which timethe cell stain was aspirated off and 500 μl of HBSS (Invitrogen, LifeTechnologies, Carlsbad, Calif.) was added to each well. The number oflive cells in each well was then quantitated using an automatedfluorescent microscope/imaging system (Universal Imaging, Downingtown PA

[0384] Results:

[0385] Certain compounds of the present invention when tested asdescribed above were active in this assay.5-Hydroxy-3,6,7-trimethyl-benzofuran-2-yl)phenyl-methanone showed 28%protection at 25 μM.

Example 9 In Vitro Cellular Inflammation Assay

[0386] A. Human Hep3B Cells—CRP assay.

[0387] Hep3B Cell Line is obtained from the American Type CultureCollection (ATCC Catalog No. HB-8064). The Hep3B cell line was derivedfrom liver tissue of an 8-year-old Black male. The cells are epithelialin morphology and produce tumors in nude mice. The cells produceα-fetoprotein, hepatitis B surface antigen, albumin, α-2-macroglobulin,α-1-antitrypsin, transferrin, plasminogen, complement C3 andβ-lipoprotein (Knowles B B, et al., Science, 1980, 209:497-499). Thiscell line has been widely used to study hepatocyte cytokine and acutephase protein release (e.g., Damtew B, et al.,1993, J Immunol150:4001-4007).

[0388] HEP3B cells are grown in Minimum Essential Medium (MEM; GIBCO)supplemented with 10% Fetal Bovine Serum (FBS; Hyclone), 1×Penicillin/Streptomycin (GIBCO, Cat #. 15140-122) and 0.1 mMnon-essential amino acids (GIBCO, Catalog No. 11140-050). Cells arethawed and transferred to warm medium according to standard methodsknown in the art. Cells are incubated in flasks at 37° C. with 5% CO₂ inan air atmosphere incubator. HEP3B growth media is changed every 2 daysuntil the cells reach 70-80% confluence (approx. 3-4 days). For assay,the cells are transferred to 96-well plates, seeded at 5000 cells perwell in culture media, and left to grow for 7 days in a 37° C. incubator(air supplemented with 5% CO₂). Media is replaced daily until assay.Test compounds are diluted into “Stimulus Buffer” (MEM medium containing0.1 mM non-essential amino acids, 1× penicillin/streptomycin, 10% FBSwith 10 ng/ml IL-1β, 20 ng/ml IL-6 and 1 μM dexamethasone. Media isremoved from the cells and is replaced with 200 μl of test dilution.Cells are returned to the incubator for three days at 37° C. CRP ELISAis then performed on supernatant from the cells, as described below.

[0389] Costar EIA/RIA plates are coated with rabbit anti-human CRP(DAKO) diluted 1:4000 in carbonate buffer (100 μl/well) for 45 minutesat 37° C. Plates are then washed 5× with CRP washing buffer (50 mMTris-HCl, 0.3M NaCl, 0.5 MI Tween-20, pH 8.0) using an automatic platewasher. Plates may be dried, covered and refrigerated until use.Supernatant (100 μl) is removed from each well of the test plates andadded to the corresponding well of a precoated ELISA plate.

[0390] 100 μl HRP-conjugated rabbit anti-human CRP (DAKO) diluted 1:500(in CRP wash buffer) is added to each well, followed by incubation for30 minutes at 37° C. Plates are washed 5× with CRP washing buffer usingthe automatic plate washer. 200 μl of 3,3′,5,5′-Tetramethyl Benzidine(TMB) liquid Substrate System (Sigma, St. Louis, Mo.) is added to eachwell, followed by incubation in the dark for 15 minutes at roomtemperature. Finally, 50 μl of 1 M H₂SO₄ is added to each well andabsorbance at 450 nm is immediately measured in a microtiterspectrophotometer.

[0391] CRP measured as above is normalized to cell count per well, usinga cell viability assay, such as the Cell Tracker Green assay. To dothis, the remainder of the medium is from the cell test plates, cellsare washed with 200 μl of pre-warmed 1× Hanks Basic Salt Solution (HBSS;GIBCO), and 100 μL of 5 μM Cell Tracker Green (Molecular Probes, Eugene,Oreg.) is added to each well. Plates are then incubated at 37° C. for 30minutes. Cells are then washed twice with prewarmed 1× HBSS. Plates areimmediately read using a Fluoroskan® flourometer with a 485excitation/538 emission filter pair.

[0392] In a CRP assay such as the one disclosed herein, compounds suchas:

[0393] 6,7-Dimethyl-3-phenyl-benzofuran-5-ol;

[0394] 1-(4-Bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone;

[0395]1-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-2-morpholin-4-yl-ethanone;

[0396] Acetic acid2-(6-hydroxy-3-methyl-[1,3]oxazinan-6-yl)-6,7-dimethyl-benzofuran-5-ylester;

[0397] 1-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone;

[0398] 3-(4-Methoxy-phenyl)-6,7-dimethyl-benzofuran-5-ol;

[0399] 4,5-Dimethyl-1,8-diphenyl-benzo[1,2-b;4,3-b′]difuran; and

[0400]1,8-Bis-(4-fluoro-phenyl)-4,5-dimethyl-benzo[1,2-b;4,3-b′]difuran;

[0401] at an EC₅₀ of between about 10 μM to 40 μM were effective atreducing CRP levels.

[0402] B. Human endothelial Cells—ELAM assy

[0403] Endothelial-Leukocyte Adhesion Molecule (ELAM), also known asE-selectin, is expressed on the surface of endothelial cells. In thisassay, lipopolysaccharide (LPS) and IL-1β are used to stimulate theexpression of ELAM; test agents are tested for their abilities to reducethis expression, in accordance with studies showing that reduction ofleukocyte adhesion to endothelial cell surface is associated withdecreased cellular damage (e.g., Takada, M., Et al., Transplantation 64:1520-25, 1997; Steinberg, J. B., et al., J. Heart Lung Trans.13:306-313, 1994).

[0404] Endothelial cells may be selected from any of a number of sourcesand cultured according to methods known in the art; including, forexample, coronary artery endothelial cells, human brain microvascularendothelial cells (HBMEC; Hess, D. C., et al., Neurosci. Lett. 213(1):37-40, 1996), or lung endothelial cells. Cells are conveniently culturedin 96-well plates. Cells are stimulated by adding a solution to eachwell containing 10 μg/ml LPS and 100 μg/ml IL-1β for 6 hours in thepresence of test agent (specific concentrations and time may be adjusteddepending on the cell type). Treatment buffer is removed and replacedwith pre-warmed Fixing Solution® (100 μl/well) for 25 minutes at roomtemperature. Cells are then washed 3×, then incubated with BlockingBuffer (PBS+2% FBS) for 25 minutes at room temperature. Blocking Buffercontaining Monoclonal E-Selectin Antibody (1:750, Sigma Catalog #S-9555)is added to each well. Plates are sealed and stored at 4° overnight.Plates are washed 4× with 160 μL Blocking Buffer per well. SecondAntibody-HRP diluted 1:5000 in Blocking Buffer is then added (100μL/well), and plates are incubated at room temperature (protected fromlight) for two hours. Plates are then washed 4× with Blocking Bufferbefore addition of 100 μL of ABTS Substrate solution at room temperature(Zymed, Catalog #00-2024). Wells are allowed to develop for 35 minutes,before measurement at 402 nm in a Fluoroskan® Reader with shake programfor 10 seconds. Positive results are recorded as a decrease in ELAMconcentration in tested wells, as compared to control wells.

[0405] In an ELAM assay, such as the one described herein, certaincompounds of the present invention at EC₅₀ in a range of 10-1000 μM, andin particular compounds such as;

[0406] 2-bromo-1-(5-acetoxy-6,7-dimethyl-benzofuran-2-yl)-ethanone;

[0407] 6,7-dimethyl-2-(4-nitro-phenyl)-benzofuran-5-ol;

[0408] 6,7-dimethyl-3-phenyl-benzofuran-5-ol;

[0409] 5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid3,7-dimethyl-octa-2,6-dienyl ester;

[0410]1-(5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-2-morpholin-4-yl-ethanone;

[0411] 3-(4-Fluoro-phenyl)-6,7-dimethyl-benzofuran-5-ol;

[0412] 3-(4-Methoxy-phenyl)-6,7-dimethyl-benzofuran-5-ol;

[0413] 3-(4-Chloro-phenyl)-6,7-dimethyl-benzofuran-5-ol;and

[0414] 5-Hydroxy-6,7-dimethyl-3-(4-nitro-phenyl)-benzofuran-2-carboxylicacid methyl ester

[0415] were able to reduce the expression of ELAM at EC₅₀ of between 10μM and 50 μM.

[0416] C. Mouse Microglial Cells-Interleukin-1.beta Assay

[0417] Materials and Equipment

[0418] A. Materials For Cell Preparation And Experiment

[0419] Mouse microgial cell line

[0420] DMEM High Glucose media (Gibco Catalog # 11965-092)

[0421] FBS (Hyclone Catalog # SH30070.03)

[0422] 100× Penicillin/Streptomycin (Gibco Catalog #15140-122).

[0423] LPS (Sigma Catalog #L2537)

[0424] Interferon-gamma (Sigma Catalog #I4777)

[0425] Cell Tracker Green (Molecular Probes Catalog #C2925)

[0426] HBSS buffer (950 ml Pyrogen-free water, 2.44 g/L MgCl2.6H20, 3.73g/L KCl, 59.58 g/L Hepes, 58.44 g/L NaCl, 1.36 g/L KH2PO4, 1.91 g/LCaCl2 .2H2O and pH to 4.5 with HCl)

[0427] Sterile 96-well plates precoated with poly-D-lysine (CorningCatalog #3665)

[0428] 96-well deep well mother plate, DyNA Block 1000 (VWR Catalog#40002-008)

[0429] B. Materials For II-1beta Elisa

[0430] Mouse IL-1beta Duo Set (R & D Systems Catalog #DY401)

[0431] Substrate Solution (R & D Systems Catalog #DY 999)

[0432] Bovine Serum Albumin fraction V (BSA V) (Sigma Catalog #A4503)

[0433] 96-well Costar EIA high binding plates (VWR Catalog #29442-302)

[0434] Plate seal (VWR Catalog #29442-310)

[0435] PBS (Irvine Scientific Catalog #9240)

[0436] Cell Culture Grade Water (Irvine Scientific Catalog #9312)

[0437] Tween 20 (Sigma Catalog #P 1379)

[0438] Sucrose (Sigma Catalog #S7903)

[0439] Sodium Azide (Sigma Catalog #S 8032)

[0440] H₂SO₄ 5N (VWR Catalog #JT 5691-2)

[0441] Experimental Preparation and Procedure:

[0442] Mouse IL-1beta Elisa:

[0443] Solutions:

[0444] Wash Buffer: PBS 1 L+500 μl Tween 20 (final 0.05%) pH 7.2-7.4.

[0445] Blocking Buffer: 500 ml PBS+5 g BSA V (1%)+25 g Sucrose (5%)+0.25g Sodium Azide (0.05%).

[0446] Reagent Diluent: 500 ml PBS+5 g BSA V (1%) pH 7.2-7.4 and filtersterilize through 0.2 μm.

[0447] Stop Solution: Make 2N sulfuric acid by adding 10 ml 5N H₂SO₄ to15 ml of dd H₂O.

[0448] Duo Set Preparations:

[0449] 1. The IL-1beta. capture antibody was reconstituted in 1 ml ofPBS to give a final concentration of 720 μg/ml, and the workingconcentration was 4 μg/ml. For coating one 96-well plate (at 100μl/well) 56 μl of the 720 μg/ml stock was diluted into 10 ml of PBS.

[0450] 2. The IL-1beta.standards were reconstituted in 0.5 ml of ReagentDiluent (70 ng/ml). For a high standard of 1 ng/ml (2wells at 100 μleach+enough for series dilution) 7.1 μl of the 70 ng/ml standard werediluted into 0.5 ml of Reagent Diluent

[0451] 3. The IL-1beta. detection antibody was reconstituted in 1 ml ofReagent Diluent to give a final concentration of 18 μg/ml and theworking concentration is 100 ng/ml. For one 96-well plate (at 100μl/well) 56 μl of the 18 μg/ml stock was diluted into 10 ml of ReagentDiluent.

[0452] IL-1.beta ELISA Procedure:

[0453] Plate Preparation:

[0454] 1. The Costar EIA Hi-binding plate was coated with captureantibody at 4 μg/ml. 56 μl of 720 μg/ml stock was taken for one plateand added to 10 ml of PBS. Each well was coated with 100 μl, and theplate was sealed and incubated overnight at room temperature.

[0455] 2. Each well was aspirated and washed 3× with Wash Buffer. Eachwell was filled to the top, dispensed, and any remaining buffer wasremoved by inverting the plate and gently blotting against clean papertowels.

[0456] 3. Non-specific binding sites were blocked by adding 300 μl ofBlocking Buffer to each well, and after sealing, incubating for at least1 hour at room temperature.

[0457] 4. After washing the plate was now ready for the samples.

[0458] Assay Procedure:

[0459] 5. 100 μl of either standard or sample were added in each well ofthe capture-coated and pre-blocked plate. The plate was sealed andincubated for 2 hours at room temperature, followed with washing as instep 2.

[0460] 6. 100 μl of the detection antibody (100 ng/ml) were added toeach well. For one 96-well plate 56 μl of the 18 μg/ml stock werediluted into 10 ml of Reagent Diluent.

[0461] 7. The plate was sealed and incubated at room temperature for 2hours, followed with washing as in step 2.

[0462] 8. 100 μl of the working dilution of Streptavidin-HRP was added,and the plate was sealed and incubated in the dark for 20 minutes atroom temperature, followed with washing as in Step 2.

[0463] 9. The fresh Substrate Solution was prepared by mixing ColorReagent A (H₂O₂) and Color Reagent B (Tetramethylbenzidine) in a 1:1ratio. 100 μl of this Substrate Solution mixture was added to each welland the plate was incubated in the dark for 20 minutes at roomtemperature.

[0464] 10. 50 μl of Stop Solution was added to each well, mixing wasensured by gently tapping.

[0465] 11. Each plate was read with the Spectramax once at 450 nm. Ifwavelength correction is available set to 540 or 570 nm.

[0466] In an IL-1 assay such as the one disclosed herein, compounds suchas:

[0467] 6,7-Dimethyl-2-(4-nitro-phenyl)-benzofuran-5-ol;

[0468] 6,7-Dimethyl-3-phenyl-benzofuran-5-ol;

[0469] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid(2-hydroxy-ethyl)-amide;

[0470] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester; and

[0471] (5-Hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone.

[0472] at an EC₅₀ of between 0.5 μM to about 40 μM were effective atreducing IL-1β levels.

Example 10 In vivo Cellular Inflammation Assay

[0473] These assays measure the ability of test compounds to prevent orreduce inflammation secondary to oxazolone or arachidonic acid.

[0474] A. Arachidonic acid. Albino male CD-1 mice, 7-9 weeks old wereused in this test. A 20% (w/v) arachidonic acid solution in acetone isprepared. Twenty microliters of the arachidonic acid solution is appliedto the dorsal left ear of the mouse. Immediately thereafter, testcompounds (20 μL in 70% ethanol/30% propylene glycol) are applied to theleft ear. The untreated right ears served as control. Mice aresacrificed by CO₂ inhalation, one hour after treatment. The left andright ears are removed and 7 mm punch biopsies taken from each. Thepunch biopsies are weighed, and the differences calculated.

[0475] B. Oxazolone. CD-1 mice are induced by applying 3% oxazolone(Sigma) (30 mg/ml prepared in corn oil:acetone) to the shaved abdomen.Five days later, the mice are challenged with 2% oxazolone (20 mg/ml) inacetone on the left ear (right ear was untreated control). One hourafter challenge, test compounds are applied to the left ear in 70%ethanol/30% propylene glycol. Animals are sacrificed 24 hours later and7 mm ear punches are removed. The ear punches are placed on a balancescale, and the difference between the untreated and treated ears isdetermined. Percent inhibition is calculated by comparing the means ofeach group to the vehicle group. (Hydrocortisone serves as a positivecontrol in this test.).

[0476] Compounds of the present invention can be tested for theirability to reduce inflammation in this model.

Example 11 6,7-Dimethyl-3-phenyl-benzofuran-5-ol

[0477]

[0478] Step 1:

[0479] A mixture of 2,3-dimethyl-1,4-dihydroquinone (1.08 g, 7.826mmol), 2-bromoacetophenone (1.45 g, 7.29 mmol), and potassium carbonate(1.78 g, 12.90 mmol) in acetone (30 mL) was stirred at room temperaturefor 3 h. The mixture was then poured into water resulting in theformation of a precipitate. The precipitate was washed with water andhexane, and dried to yield a mixture of mono and bis products.Purification by silica gel column eluting with 30% EtAOc in hexane,2-(4-Hydroxy-2,3-dimethyl-phenoxy)-1-phenyl-ethanone (0.65 g) and2-[2,3-Dimethyl-4-(2-oxo-2-phenyl-ethoxy)-phenoxy]-1-phenyl-ethanone(0.5 g).

[0480] Step 2:

[0481] A mixture of 2-(4-hydroxy-2,3-dimethyl-phenoxy)-1-phenyl-ethanone(1.2 g, 4.70 mmol) and polyphosphoric acid (ca. 100 mg) in xylene (10mL) was stirred at 150° C. for 5 h. The mixture was poured into water,extracted with ethylacetate, washed and dried to give6,7-dimethyl-3-phenyl-benzofuran-5-ol (560 mg) as a light brown solid.¹H NMR (CDCl₃, 300 MHz) δ: 7.71 (s, 1H), 7.60-7.20 (m, 5H), 7.06 (s,1H), 4.76 (s, 1H, OH), 2.47, 2.29 (2s, 6H) ppm. ¹³C NMR (CDCl_(3,) 75MHz) δ: 150.43, 150.30, 141.15, 132.58, 129.00, 127.32, 123.48, 122.13,121.31, 120.59, 102.25, 12.31, 11.95 ppm. MS (m/z): 239 (M+H⁺).

[0482] Similarly by following the procedure described above butreplacing bromoacetophenone with the appropriate substitutedbromoacetophenones the following compounds were obtained:

[0483] 3-(4-Fluoro-phenyl)-6,7-dimethyl-benzofuran-5-ol; ¹H NMR (CDCl₃,300 MHz) δ: 7.65 (m, 1H), 7.50 (m, 2H), 7.10-6.95 (m, 3H), 4.90 (s, 1H,OH), 2.44, 2.31 (2s, 6H) ppm. ¹³C NMR (CDCl₃, 75 MHz) δ: 163.79, 160.53,150.37, 150.30, 140.97, 128.90, 128.79, 128.56, 128.52, 123.44, 121.41,121.24, 120.78, 116.06, 115.78, 102.04, 12.30, 11.96 ppm. ¹⁹F NMR(CDCl₃, 300 MHz, TFA as reference) δ: −115.31 ppm. MS (m/z): 257 (M+H⁺).

[0484] 3-(4-Methoxy-phenyl)-6,7-dimethyl-benzofuran-5-ol ¹H NMR (CDCl₃,300 MHz) δ: 7.64 (s, 1H), 7.50 (dd, J=2.1, 6.7 Hz, 2H, 2H), 7.00 (m,3H), 4.83 (s, 1H, OH), 3.84 (s, 3H), 2.46, 2.28 (2s, 6H) ppm. ¹³C NMR(CDCl₃, 75 MHz) δ: 158.92, 150.33, 150.23, 140.52, 128.47, 125.02,123.69, 121.70, 121.25, 120.49, 114.45, 102.20, 55.47, 12.30, 11.95 ppm.MS (m/z): 269 (M+H⁺).

[0485] 3-(4-Chloro-phenyl)-6,7-dimethyl-benzofuran-5-ol. ¹H NMR (CDCl₃,300 MHz) δ: 7.72 (s, 1H), 7.53 (d, J=8.6 Hz, 2H), 7.42 (d, J=8.6 Hz,2H), 7.02 (s, 1H), 4.86 (s, 1H, OH), 2.47, 2.30 (2s, 6H) ppm. ¹³C NMR(CDCl₃, 75 MHz) δ: 150.41, 141.25, 133.04, 131.06, 129.17, 128.49,123.17, 121.45, 121.12, 120.79, 101.99, 12.30, 11.94 ppm. MS (m/z): 273,275 (M+H⁺).

Example 12 4,5-Dimethyl-1,8-diphenyl-benzo[1,2-b;4,3-b′]difuran

[0486]

[0487] A mixture of2-[2,3-Dimethyl-4-(2-oxo-2-phenyl-ethoxy)-phenoxy]-1-phenyl-ethanone(480 mg, 1.28 mmol) synthesized as described in Example 11, an excess ofbromoacetophenone, and polyphosphoric acid (PPA, 100 mg) in xylene (10mL) was stirred at 150° C. for 10 h. The mixture was then poured intowater, extracted with EtOAc, the organic layer was washed with water andbrine, dried over MgSO₄ and concentrated. The residue was purified bysilica gel column eluting with 10-20% EtOAc in hexane to give 190 mg of4,5-dDimethyl-1,8-diphenyl-benzo[1,2-b;4,3-b′]difuran (250 mg). ¹H NMR(CDCl₃, 300 MHz) δ: 7.60 (s, 2H), 7.05 (m, 4H), 6.90 (m, 2H), 6.85 (m,4H), 2.60 (s, 6H) ppm. ^(13C)NMR (CDCl₃, 75 MHz) δ: 152.57, 141.48,133.19, 128.42, 127.52, 126.87, 124.05, 117.40, 116.29, 11.80 ppm. MS(m/z): 339 (M+H⁺).

[0488] Similarly by following the procedure of Example 12, the followingcompounds were obtained:

[0489]1,8-Bis-(4-fluoro-phenyl)-4,5-dimethyl-benzo[1,2-b;4,3-b′]difuran; . ¹HNMR (CDCl₃, 300 MHz) δ: 7.56 (s, 2H), 7.05 (m, 4H), 6.60 (m, 4H), 2.60(s, 6H) ppm. ¹³C NMR (CDCl₃, 75 MHz) δ: 163.91, 160.66, 152.51, 141.44,130.18, 129.16, 129.12, 122.77, 117.59, 116.27, 114.65, 114.36, 11.78ppm. ¹⁹F NMR (CDCl₃, 300 MHz, TFA as reference) δ: −116.57 ppm. MS(m/z): 375 (MH⁺).

[0490]1,8-Bis-(4-methoxy-phenyl)-4,5-dimethyl-benzo[1,2-b;4,3-b′]difuran). ¹HNMR (CDCl₃, 300 MHz) δ: 7.56 (s, 2H), 6.95 (d, J=8.7 Hz, 4H), 6.90 (m,2H), 6.43 (d, J=8.7 Hz, 4H), 3.75 (s, 6H), 2.60 (s, 6H) ppm. ¹³C NMR(CDCl₃, 75 MHz) δ: 158.49, 152.39, 141.07, 129.78, 125.51, 123.5,117.24, 116.74, 112.98, 54.94, 11.78 ppm. MS (m/z): 399 (M+H⁺).

Example 13 (5-Hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone

[0491]

[0492] Step 1:

[0493] To a suspension of 2,3-dimethyl-1,4-dihydroquinone (11.58 g) andtriethylamine (25 mL) in dichloromethane (100 mL) was slowly addedacetic anhydride (16 mL). Then the mixture was stirred at roomtemperature for 2 h until the solid was dissolved. The mixture waswashed with water, dried over MgSO4, and concentrated to dryness to givea solid. The solid was washed with hexane and ether to give 15.40 g ofthe 1,4 diacetate derivative of the starting material.

[0494] Step 2:

[0495] A suspension of the diacetate from Step 1 (5.08 g) in borontrifluoride acetic acid complex (15 mL) was stirred at 110° C. for 1 h.After cooling, the mixture was poured into ice, and the mixture wasextracted with dichloromethane. The organic layer was washed with waterand dried. After evaporation, the residue was recrystallized fromEtOAc-hexane to give 4.37 g of1-(2-hydroxy-5-acetoxy-3,4-dimethyl-phenyl)-ethanone.

[0496] Step 3:

[0497] The phenylethanone of Step 2 (500 mg) was dissolved in MeOH (20mL), and potassium carbonate (1 eq.) was added followed by water (1 mL).The mixture was stirred at room temperature for 1 h, and then was pouredinto water. The solution was acidified with HCl and a precipitate wasformed. The precipitate was collected and air dried to give about 350 mgof 1-(2,5-dihydroxy-3,4-dimethyl-phenyl)-ethanone.

[0498] Step 4:

[0499]1-(2,5-Dihydroxy-3,4-dimethyl-phenyl)-ethanone of Step 3 (275 mg)was dissolved in dichloromethane (20 mL) and 3,4-dihydro-2H-pyran (0.2mL) was added followed by pyridinium p-toluenesulfonate (PPTS) (30 mg).The mixture was stirred at room temperature for 4 h. The mixture wasdried over MgSO₄ and purified on silica gel column eluting with 30%EtOAc in hexane to give 397 mg of1-[2-hydroxy-3,4-dimethyl-5-(tetrahydro-pyran-2-yloxy)-phenyl]-ethanoneas a yellow solid.

[0500] Step 5

[0501] A mixture of the tetrahydropyran ether from Step 4 (250 mg),2-bromo-1-phenyl-ethanone (250 mg), and potassium carbonate (300 mg) inDMF was stirred at room temperature for 3 h. The mixture was poured intowater and extracted with ethyl acetate; the organic layer was dried andevaporated. Purification on a silica gel column eluting with 30% EtOAcin hexane gave 260 mg of a colorless solid which was then dissolved inDMF followed by the addition of cesium carbonate (2 eq.). The mixturewas stirred overnight at room temperature, then poured into water andextracted with ethylacetate, the organic layer was dried and evaporated.The crude product was dissolved in MeOH with dil. HCl and stirred for 1h. It was then poured into water and extracted with ethylacetate; theorganic layer was washed, dried and evaporated. Purification on a silicagel column eluting with 30% EtOAc in hexane gave(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone (187 mg).¹H NMR (CDCl3 with MeOH-d4, 300 MHz)δ: 8.10(m, 2H), 7.50(m, 3H), 6.88(s,1H), 2.55, 2.43, 2.30 (3s, 9H) ppm. ¹³C NMR (CDCl₃ with MeOH-d4, 75 MHz)δ:186.58, 152.26, 149.41, 148.23, 138.47, 132.81, 130.08, 128.61,128.38, 127.37, 126.55, 121.64, 101.68, 101.63, 12.72, 12.51, 10.53 ppm.MS: 281 (M+H⁺).

Example 14 1-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone

[0502]

[0503] A mixture of1-[6,7-dimethyl-5-(tetrahydro-pyran-2-yloxy)-benzofuran-2-yl]-ethanone(1.1 g) (prepared from 2,5-dihydroxy-3,4-dimethyl-benzaldehyde followingthe procedure of Steps 3, 4 and 5 of Example 13) and conc. HCl (10drops) in MeOH (20 mL) was stirred at room temperature for 2 h. Waterwas poured into the mixture and left to precipitate overnight in therefrigerator. The precipitate was collected, washed with hexane anddried to give 1-(5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone as alight brown solid (490 mg). ¹H NMR (CDCl₃, 300 MHz) δ: 7.38(s, 1H), 6.89(s, 1H), 4.88 (1H, OH), 2.60, 2.50, 2.31 (3s, 9H) ppm. ¹³C NMR (CDCl₃,75 MHz) δ: 192.76, 155.45, 155.13, 153.72, 130.29, 127.27, 124.75,117.44, 106.18, 29.30, 15.56, 15.28 ppm. MS (m/z): 205 (M+H⁺).

Example 15 1-(3-Bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone

[0504]

[0505] To a solution of1-[6,7-dimethyl-5-(tetrahydro-pyran-2-yloxy)-benzofuran-2-yl]-ethanone(200 mg) in chloroform (20 mL) was added bromine (120 mg). After 1 h,the solution was washed with water and dried over MgSO₄ andconcentrated. The residue was purified by silica gel column eluting with10% hexane in DCM to give1-(3-Bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone, as a yellowsolid. ¹H NMR (CDCl₃, 300 MHz) δ: 7.38(s, 1H), 5.55 (s, 1H, OH), 2.60,2.50, 2.31 (3s, 9H) ppm. MS (m/z): 283, 285 (M+H⁺).

Example 16 2-Bromo-1-(5-acetoxy-6,7-dimethyl-benzofuran-2-yl)-ethanone

[0506]

[0507] 1-(5-Acetoxy-6,7-dimethyl-benzofuran-2-yl)-ethanone (83 mg)(prepared from 1-(5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-ethanone fromExample 14 with acetanhydride and pyridine), was dissolved in chloroform(10 mL). Bromine (50 mg) was added and the solution was stirred at 70°C. for 10 min. When the color of bromine disapeared, the mixture wasevaporated to dryness. The residue was purified by silica gel columneluting with 30% EtOAc in hexane to give 85 mg of2-bromo-1-(5-acetoxy-6,7-dimethyl-benzofuran-2-yl)-ethanone as a whitesolid. ¹H NMR (CDCl₃, 300 MHz) δ: 7.58 (s, 1H), 7.26 (s, 1H), 4.43(s,2H), 2.51, 2.37, 2.21(3s, 9H) ppm. ¹³C NMR (CDCl₃, 75 MHz) δ: 182.55,170.13, 153.71, 150.74, 146.81, 131.21, 124.51, 122.92, 115.15, 113.28,30.69, 21.27, 13.54, 12.80 ppm.

Example 17 Acetic Acid6,7-dimethyl-2-(2-morpholin-4-yl-acetyl)-benzofuran-5-yl ester

[0508]

[0509] A solution of2-bromo-1-(5-acetoxy-6,7-dimethyl-benzofuran-2-yl)-ethanone (47 mg) fromExample 16, morpholine (15 mg), and potassium carbonate (25 mg) inacetone (10 mL) was stirred at room temperature for 30 min. It was thenevaporated to dryness, and the residue was purified by silica gel columneluting with 5% MeOH in DCM to give 43 mg of acetic acid6,7-dimethyl-2-(2-morpholin-4-yl-acetyl)-benzofuran-5-yl ester as anoil. Conversion into the HCl salt gave a yellow solid. ¹H NMR (CDCl₃,300 MHz) δ: 7.60 (s, 1H), 7.20 (s, 1H), 3.78(s+t, 6H), 2.64(m, 4H),2.50(s, 3H), 2.40(s, 3H), 2.20(s, 3H) ppm. ¹³C NMR (CD₃OD, 75 MHz) (asHCl salt) δ: 180.94, 170.31, 153.65, 150.33, 147.19, 131.94, 124.33,122.34, 116.04, 113.59, 63.74, 60.89, 53.28, 19.64, 12.18, 12.22 ppm.M/S 332 (M+H⁺).

Example 181-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-2-morpholin-4-yl-ethanone

[0510]

[0511] A solution of acetic acid6,7-dimethyl-2-(2-morpholin-4-yl-acetyl)-benzofuran-5-yl ester (23 mg),prepared as in Example 17, in MeOH (10 mL) was stirred while sodiumbicarbonate (10 mg) was added. Then the mixture was stirred at RTovernight, followed by evaporation to dryness. The residue was purifiedby silica gel column, eluting with 5% MeOH in dichloromethane to give 11mg of1-(5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-2-morpholin-4-yl-ethanone asan oil. Conversion into HCl salt gave a yellow solid. ¹H NMR (CD₃OD, 300MHz) δ:: 7.60 (s, 1H), 7.20 (s, 1H), 3.78(m, 6H), 2.64(m, 4H), 2.50(s,3H), 2.40(s, 3H) ppm. ¹³C NMR (CDCl₃, 75 MHz)δ: 187.98, 170.20, 153.31,152.29, 146.58, 130.44, 124.53, 122.80, 114.36, 113.15, 64.69, 53.79,21.28, 13.47, 12.86 ppm. M/S 290 (M+H⁺).

Example 192-(1-Hydroxy-2-morpholin-4-yl-ethyl)-6,7-dimethyl-benzofuran-5-ol

[0512]

[0513] A solution of acetic acid6,7-dimethyl-2-(2-morpholin-4-yl-acetyl)-benzofuran-5-yl ester (35 mg),prepared as in Example 17, in MeOH (10 mL) was stirred while NaBH4 (40mg) was added, and the mixture was stirred at room temperature for anadditional 4 h. The mixture was poured into water, and extracted withethylacetate. The organic layer was dried over MgSO₄ and evaporated. Theresidue was purified by silica gel column eluting with 5% MeOH in DCM togive 21 mg of2-(1-hydroxy-2-morpholin-4-yl-ethyl)-6,7-dimethyl-benzofuran-5-ol as anoil. Conversion into HCl salt gave a yellow solid. ¹H NMR (CD₃OD, 300MHz) δ: 6.77 (s, 1H), 6.67 (s, 1H), 5.29 (dd, J=10.4, 3.3 Hz, 1H),4.04(m, 2H), 3.85(q, 2H), 3.70-3.50 (m, 4H), 3.30(m, 2H), 2.40(s, 3H),2.20(s, 3H) ppm. ¹³C NMR (CD₃OD, 75 MHz) (as HCl salt) δ: 154.72,151.88, 149.40, 125.02, 121.67, 120.21, 104.51, 102.50, 63.77, 63.69,61.70, 60.19, 53.82, 51.28, 11.24, 11.10 ppm. M/S 292 (M+H⁺).

Example 202-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-4-methyl-morpholin-2-ol

[0514]

[0515] A mixture of2-bromo-1-(5-acetoxy-6,7-dimethyl-benzofuran-2-yl)-ethanone (30 mg),methylaminoethanol (7 mg), and potassium carbonate (20 mg) in acetone(10 mL) was stirred at RT for 1 h. The mixture was poured into water andextracted with ethylacetate. The organic layer was dried and evaporatedfollowed by purification by silica gel column chromatography elutingwith 5% MeOH in DCM to give 20 mg of2-(5-hydroxy-6,7-dimethyl-benzofuran-2-yl)-4-methyl-morpholin-2-ol as anoil, and was converted into the HCl salt. The NMR showed an equilibriumof the hemiketal and ketone form. ¹H NMR (CDCl₃, 300 MHz) δ: 7.51, 7.26(2s, combined, 1 H), 7.06, 6.87 (2s, combined 1H), 4.2-3.7 (m, 2H),3.2-2.8 (m, 4H), 2.5-2.0 (m, 12 H) ppm. ¹³C NMR (CDCl₃, 75 MHz) δ:189.26, 170.43, 170.18, 156.79, 152.53, 145.71, 125.79, 125.39, 124.46,121.73, 113.88, 113.14, 111.52, 104.28, 92.48, 63.55, 62.62, 61.11,59.68, 54.75, 46.37, 21.30, 13.48, 13.02, 12.90 ppm. M/S 320 (M+H⁺).

Example 21 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methylester

[0516]

[0517] Step 1:

[0518] 2,5-Dihydroxy-3,4-dimethyl-benzaldehyde (2.0 g) was dissolved indichloromethane (50 mL) and 3,4-dihydro-2H-pyran (1.5 g) was addedfollowed by addition of p-toluenesulfonic acid monohydrate (200 mg). Thesolution was stirred at room temperature for 1 hour and quenched byadding sodium bicarbonate solution (1 mL). Then the dichloromethanesolution was dried over MgSO₄ and concentrated. The residue was purifiedby silica gel column eluting with hexane and ethyl acetate (8:2) to give1.5 g of2-hydroxy-3,4-dimethyl-5-(tetrahydro-pyran-2-yloxy)-benzaldehydeproduct.

[0519] Step 2

[0520] A mixture of2-hydroxy-3,4-dimethyl-5-(tetrahydro-pyran-2-yloxy)-benzaldehyde (150mg) from Step 1, methyl bromoacetate (180 mg) and potassium carbonate(200 mg) in DMF (10 mL) was stirred at room temperature for 2 hours.Then the solution was poured into water and extracted with ethylacetate.The ethyl acetate was washed with water and brine, dried over MgSO₄ andconcentrated. The residue was purified by silica gel column eluting withhexane and ethyl acetate (9:1) to give 100 mg of[6-formyl-2,3-dimethyl-4-(tetrahydro-pyran-2-yloxy)-phenoxy]-acetic acidmethyl ester, which was then dissolved in DMF (15 mL). To this solutionwas added cesium carbonate (200 mg) and the mixture was stirred at roomtemperature overnight. Then the solution was poured into water andextracted with ethylacetate. The organic layer was washed with water andbrine, dried over MgSO₄ and concentrated to give6,7-dimethyl-5-(tetrahydro-pyran-2-yloxy)-benzofuran-2-carboxylic acidmethyl ester. The tetrahydropyranyl ether was dissolved in methanol,p-toluenesulfonic acid monohydrate (20 mg) was added, and the solutionwas stirred at room temperature for an additional 30 min. After theaddition of a small amount of NaHCO₃, the methanol was evaporated. Theresidue was purified by silica gel column chromatography, eluting withhexane and ethyl acetate (7:3) to give 30 mg of5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester. ¹H NMR(CDCl₃, 300 MHz) δ: 7.39 (s, 1H), 6.87 (s, 1H), 4.75 (s, 1H), 3.95 (s,3H), 2.50, 2.26 (2s, 6H) ppm.

Example 22 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester

[0521]

[0522] A mixture of acid (50 mg),2-[2-(2-methoxy-ethoxy)-ethoxy]-ethanol (100 μL), DCC (120 mg), DMAP (50mg) in DCM (20 mL) was stirred at RT for overnight. The mixture waspoured into water and extracted with ethylacetate. The organic layer waswashed with water and brine, dried over MgSO₄ and concentrated. Theresidue was purified by silica gel column chromatopraphy eluting with40%EtOAc in hexane to give the tetrahydropyranyl ether of5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester (40 mg). This intermediatewas dissolved in MeOH, diluted HCl was added, and the mixture wasstirred for 30 min. The mixture was poured into water and extracted withethylacetate. The organic layer was washed with water and brine, driedover MgSO4 and concentrated. Purification by silica gel column elutingwith 2% MeOH in DCM gave 30 mg of5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester. ¹HNMR (CDCl₃, 300 MHz) δ:7.27(s, 1 H), 6.84(s, 1H), 6.30(br., 1H) 4.50(m, 2H), 3.87(m, 2H),3.71-3.50(m, 8H), 3.35(s, 3H), 2.42(s, 3H), 2.26(s, 3H) ppm. ¹³CNMR(CDCl₃, 75 MHz) δ: 159.33, 150.91, 150.11, 143.97, 124.82, 123.33,121.14, 102.70, 71.04, 70.23, 70.15, 70.11, 68.76, 63.74, 58.56, 11.86,11.81 ppm. MS: 375.1 (M+H⁺).

[0523] Similarly by following the procedure described above butreplacing 2-[2-(2-methoxy-ethoxy)-ethoxy]-ethanol with the appropriatealcohol the following compounds were prepared:

[0524] Geraniol gave 5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid3,7-dimethyl-octa-2,6-dienyl ester; ¹HNMR (CDCl₃, 300MHz) δ: 7.54(s,1H), 7.07(s, 1H), 5.48(m, 1H), 5.36(s, 1H), 5.09(m, 1H), 4.87(m, 2H),2.49(s, 3H), 2. 28(s, 3H), 2.08(m, 4H), 1.80, 1.60, 1.39 (3s, 9H) ppm.¹³CNMR (CDCl₃, 75 MHz) δ: 159.67, 150.50, 150.28, 144.67, 142.55,131.49, 124.38, 123.53, 123.27, 121.39, 117.48, 113.59, 102.87, 61.79,39.14, 25.83, 25.26, 17.28, 16.19, 11.89, 11.94 ppm. MS: 343(M+H⁺).

[0525] 2-Aminoethanol gave5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid(2-hydroxy-ethyl)-amide; 1HNMR(DMSO-d6, 300 MHz) δ: 9.3 (br., 1H),8.42(s, 1H), 7.25(s, 1H), 6.83(s, 1H), 4.77(br., 1H) 3.47(m, 4H),2.37(s, 3H), 2.11(s, 3H) ppm. ¹³CNR(DMSO-d6, 75 MHz) δ: 158.90, 151.77,148.24, 147.89, 123.98, 123.77, 120.63, 109.69, 102.62, 59.44, 12.20,11.95 ppm. MS 250 (M+H+).

[0526] 2-(2-Hydroxy-ethylamino)-ethanol gave5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acidbis-(2-hydroxy-ethyl)-amide. ¹HNMR (CD₃OD, 300 MHz) δ: 7.26(s, 1H),6.83(1H), 3.90-3.70(m, 8H), 2.40(s, 3H), 2.23(s, 3H )ppm. ¹³CNMR (CD₃OD,75MHz) δ: 161.71, 151.76, 148.60, 147.35, 123.77, 123.25, 119.93,112.09, 101.73, 60.29, 58.78, 51.76, 50.27, 10.49, 10.43 ppm. MS 294(M+H⁺).

[0527] Morpholine gave(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-morpholin-4-yl-methanone; ¹HNMR(DMSO-d₆-D₂O, 300 MHz) δ: 7.22(s, 1H), 5.88(1H), 3.65(m, 8H), 2.34(s,3H), 2.15(s, 3H) ppm. MS 276 (M+H⁺).

Example 23 2-Hydroxymethyl-6,7-dimethyl-benzofuran-5-ol

[0528]

[0529] 5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester(231 mg) prepared as in Example 2, was dissolved in THF and lithiumaluminum hydride (93 mg) was added. The mixture was stirred at roomtemperature for 4 h. Then the solution was poured into water andextracted with ethylacetate. The ethyl acetate was washed with water andbrine, dried over MgSO₄ and concentrated. Purification by silica gelcolumn eluting with 50% EtOAc in hexane gave 89 mg of2-Hydroxymethyl-6,7-dimethyl-benzofuran-5-ol. ¹HNMR (CD₃OD, 300 MHz) δ:6.75, 1H), 6.47(s, 1H), 4.61 (s, 2H), 2.37(s, 3H), 2.20(s, 3H) ppm.¹³CNMR (CD₃OD, 75 MHz) δ: 155.87, 150.51, 148.68, 124.69, 119.93,119.20, 103.01, 101.62, 56.32, 10.43, 10.29 ppm. MS: 175(M−OH)⁺.

Example 24 6,7-Dimethyl-2-(4-nitro-phenyl)-benzofuran-5-ol

[0530]

[0531] A mixture of2-hydroxy-3,4-dimethyl-5-(tetrahydro-pyran-2-yloxy)-benzaldehyde (200mg) prepared as in Example 21, p-nitrobenzyl bromide (200 mg) andpotassium carbonate (220 mg) in DMF (10 mL) was stirred overnight atroom temperature. Then the solution was poured into water and extractedwith ethylacetate. The ethylacetate was washed with water and brine anddried over MgSO₄ and concentrated. The residue was purified by silicagel column eluting with hexane: ethylacetate (8:2) to give 242 mg of thetetrahydropyranyl ether of6,7-Dimethyl-2-(4-nitro-phenyl)-benzofuran-5-ol. A mixture of the ether(240 mg) and NaOMe (17 mg) in DMF (10 mL) was stirred at 70° C. for 2 h.Then the solution was poured into water and extracted with ethylacetate.The organic layer was washed with water and brine, dried over MgSO4 andconcentrated. The residue was dissolved in MeOH and p-toluenesulfonicacid monohydrate (20 mg) was added and the solution was stirred at roomtemperature for 1.5 h. After the addition of a small amount of NaHCO₃,the methanol was evaporated, and the residue was purified by silica gelcolumn chromatography eluting with hexane and ethyl acetate (7:3) togive 170 mg of 6,7-dimethyl-2-(4-nitro-phenyl)-benzofuran-5-ol. 1H NMR(CDCl₃-DMSO-d₆, 300 MHz) δ: 9.16 (s, 1H), 8.28 (d, J=8.1 Hz, 2H), 8.06(d, J=8.1 Hz, 2H), 7.46 (s, 1H), 6.87 (s, 1H), 2.44, 2.18 (2s, 6H) ppm.¹³C NMR (CDCl3-DMSO-d6, 75 MHz) δ: 152.87, 152.57, 149.68, 147.15,137.19, 126.16, 125.64, 125.07, 123.56, 120.66, 107.03, 103.13, 12.92,12.84 ppm.

[0532] Similarly following the procedure described above, the followingcompound was prepared:4-(5-Hydroxy-6,7-dimethyl-benzofuran-2-yl)-benzonitrile; ¹H NMR(DMSO-d₆, 300 MHz) δ: 9.24 (s, 1H), 8.00 (d, J=8.0 Hz, 2H), 7.90 (d,J=8.0 Hz, 2H), 7.47 (s, 1H), 6.87 (s, 1H), 2.41, 2.18 (2s, 6H) ppm. ¹³CNMR (DMSO-d₆, 75 MHz) δ: 152.92, 152.82, 149.36, 135.22, 133.75, 126.19,125.50, 123.17, 120.75, 119.72, 110.80, 106.30, 103.10, 12.95, 12.84 ppm.

Example 25 5-Hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde

[0533]

[0534] A mixture of2-hydroxy-3,4-dimethyl-5-(tetrahydro-pyran-2-yloxy)-benzaldehyde (300mg), bromoacetaldhyde dimethyl acetal (300 mg) and potassium carbonate(330 mg) in DMF (10 mL) was stirred at 140° C. for 2 h. Then thesolution was poured into water and extracted with ethylacetate. Theorganic layer was washed with water and brine, dried over MgSO4 andconcentrated. The residue was purified by silica gel columnchromatography eluting with hexane:ethylacetate (8:2) to give 102 mg ofthe tetrahydropyranyl ether of5-hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde . A solution of thisintermediate (100 mg) in acetic acid (15 mL) was refluxed for 2 h. Thenacetic acid was evaporated and the residue was purified on silica gelcolumn chromatography eluting with 30% ethyl acetate in hexane to give70 mg of 5-hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde. Furtherpurification by silica gel eluting with dichloromethane and ethylacetate (9:1) gave a pure product (36 mg). ¹H NMR (DMSO-d₆, 300 MHz) δ:9.75 (s, 1H), 9.55 (s, 1H), 7.79 (s, 1H), 7.00 (s, 1H), 2.39 (s, 3H),2.20 (s, 3H) ppm. ¹³C NMR (DMSO-d6, 75 MHz) δ: 181.06, 181.02, 153.46,152.89, 150.56, 128.00, 124.47, 121.72, 120.79, 104.14, 13.34, 12.84ppm.

[0535] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. All patents and publications cited above arehereby incorporated by reference.

We claim:
 1. A compound represented by Formula II:

wherein: R⁶ is: hydrogen, optionally substituted (C₁-C₆)-alkyl,optionally substituted (C₂-C₁₀)-alkenyl, optionally substituted aryl,(optionally substituted (C₁-C₆)-alkoxy)carbonyl, or halogen; R⁷ and R⁸are independently selected from optionally substituted (C₁-C₆)-alkyl,optionally substituted (C₂-C₁₀)-alkenyl, or optionally substituted(C₃-C₈)-cycloalkyl; R⁹ is: optionally substituted aryl, (optionallysubstituted(C₁-C₆)-alkyl)carbonyl, (optionally substitutedaryl)carbonyl, (optionally substituted heterocyclyl)carbonyl,(optionally substituted heterocyclylalkyl)carbonyl, (optionallysubstituted (C₁-C₆)-alkoxy)carbonyl, (optionally substituted(C₂-C₁₀)-alkenyloxy)carbonyl, (optionally substituted amino)carbonyl,carboxy, formyl, or hydroxy(optionally substituted)(C₁-C₆)-alkyl; R¹⁰is: (C₁-C₆)-alkyl, (C₂-C₁₀)-alkenyl, or amino; and R′ is: hydrogen,(C₁-C₆)-alkyl, (C₁-C₆)-alkylcarbonyl, phosphoryl, or polyalkoxy; or R′and R⁶ with the atoms to which they are attached form an optionallysubstituted ring; with the proviso that the compound is not(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone or3-amino-5-hydroxy-4,6,7-trimethyl-benzofuran-2-carboxylic acid ethylester; and single stereoisomers, mixtures of stereoisomers, and thepharmaceutically acceptable salts thereof.
 2. The compound of claim 1,wherein R⁷ and R⁸ are (C₁-C₆)-alkyl and R⁶ is hydrogen.
 3. The compoundof claim 2, wherein R¹⁰ is (C₁-C₆)-alkyl, and R′ is hydrogen.
 4. Thecompound of claim 1, wherein R⁹ is phenylcarbonyl and wherein saidphenyl group is unsubstituted or substituted with one or moresubstituents selected from alkyl, alkenyl, alkoxy, hydroxy,hydroxyalkyl, haloalkyl, (optionally substituted alkoxy)carbonyl,carboxy, nitro, halo, and cyano.
 5. The compound of claim 2, wherein R¹⁰is amino and R⁹ is (C₁-C₆)-alkoxycarbonyl.
 6. A pharmaceuticalcomposition comprising a compound of claim 1 admixed with an acceptableexcipient.
 7. A method of treatment for a mammal suffering from acondition characterized by oxidative stress, comprising administering atherapeutically effective amount of a compound of claim
 1. 8. The methodof treatment for a mammal suffering from a condition characterized byoxidative stress, comprising administering a therapeutically effectiveamount of a pharmaceutical composition of claim
 6. 9. The method ofclaim 7, wherein the condition is selected from stroke, cerebralischemia, retinal ischemia, myocardial infarction, chronic heartfailure, post-surgical cognitive dysfunctions, peripheral neuropathy,spinal cord injury, head injury, and surgical trauma.
 10. The method ofclaim 7, wherein the condition involves inflammatory or autoimmunecomponents.
 11. The method of claim 10, wherein the inflammatorycondition is a dermatologic condition.
 12. A method of treatment for amammal suffering from a condition characterized by mitochondrialdysfunction or neurodegeneration, comprising administering atherapeutically effective amount of a compound of claim
 1. 13. Themethod of claim 12, wherein the condition is selected from Alzheimer'sdisease, Parkinson's disease, Friedreich's ataxia, cerebellar ataxias,Leber's hereditary optic neuropathy, epilepsy, and myodegenerativedisorders.
 14. The method of claim 13, wherein the condition isepilepsy.
 15. The method of claim 13, wherein the condition isParkinson's disease.
 16. The method of claim 13, wherein the conditionis Friedreich's ataxia.
 17. A method of protecting cellularmitochondrial function against a toxic insult with compounds of claim 1.18. The method of claim 17, wherein said cellular mitochondrial functionis in a neuronal cell.
 19. The method of claim 18, wherein said neuronalcell is dopaminergic cell.
 20. The method of claim 19, wherein saiddopaminergic cells are in the neurons of the substantia nigra-parscompacta.
 21. A pharmaceutical composition comprising(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone admixedwith an acceptable excipient.
 22. A method of treatment for a mammalsuffering from a condition characterized by oxidative stress, comprisingadministering a therapeutically effective amount of(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone.
 23. Themethod of treatment for a mammal suffering from a conditioncharacterized by oxidative stress, comprising administering atherapeutically effective amount of a pharmaceutical composition ofclaim
 21. 24. The method of claim 22, wherein the condition is selectedfrom stroke, cerebral ischemia, retinal ischemia, myocardial infarction,chronic heart failure, post-surgical cognitive dysfunctions, peripheralneuropathy, spinal cord injury, head injury, and surgical trauma. 25.The method of claim 22, wherein the condition involves inflammatory orautoimmune components.
 26. The method of claim 25, wherein theinflammatory condition is a dermatologic condition.
 27. A method oftreatment for a mammal suffering from a condition characterized bymitochondrial dysfunction or neurodegeneration, comprising administeringa therapeutically effective amount of(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone.
 28. Themethod of claim 27, wherein the condition is selected from Alzheimer'sdisease, Parkinson's disease, Friedreich's ataxia, cerebellar ataxias,Leber's hereditary optic neuropathy, epilepsy, and myodegenerativedisorders.
 29. The method of claim 28, wherein the condition isepilepsy.
 30. The method of claim 28, wherein the condition isParkinson's disease.
 31. The method of claim 28, wherein the conditionis Friedreich's ataxia.
 32. A method of protecting cellularmitochondrial function against a toxic insult with(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl)-phenyl-methanone.
 33. Themethod of claim 32, wherein said cellular mitochondrial function is in aneuronal cell.
 34. The method of claim 33, wherein said neuronal cell isdopaminergic cell.
 35. The method of claim 34, wherein said dopaminergiccells are in the neurons of the substantia nigra-pars compacta.
 36. Amethod of treatment for a mammal suffering from a conditioncharacterized by mitochondrial dysfunction or neurodegeneration,comprising administering a therapeutically effective amount of acompound of Formula I:

wherein: R′ is: hydrogen, optionally substituted (C₁-C₆)-alkyl,optionally substituted (C₂-C₁₀)-alkenyl, optionally substituted aryl,(optionally substituted (C₁-C₆)-alkoxy)carbonyl, or halogen; R² and R³are independently selected from optionally substituted (C₁-C₆)-alkyl,optionally substituted (C₂-C₁₀)-alkenyl, or optionally substituted(C₃-C₈)-cycloalkyl; R⁴ is: hydrogen, optionally substituted aryl,(optionally substituted(C₁-C₆)-alkyl)carbonyl, (optionally substitutedaryl)carbonyl, (optionally substituted heterocyclyl)carbonyl,(optionally substituted heterocyclylalkyl)carbonyl, (optionallysubstituted (C₁-C₆)-alkoxy)carbonyl, (optionally substituted(C₂-C₁₀)-alkenyloxy)carbonyl, (optionally substituted amino)carbonyl,carboxy, formyl, or hydroxy(optionally substituted)(C₁-C₆)-alkyl; R⁵ is:hydrogen, (C₁-C₆)-alkyl, (C₂-C₁₀)-alkenyl, (optionally substitutedalkoxy)carbonyl, carboxy, (optionally substituted amino)carbonyl, oroptionally substituted aryl; provided that one of R⁴ or R⁵ is hydrogen,and that when R⁴ is hydrogen R⁵ is not hydrogen, and when R⁵ is hydrogenR⁴ is not hydrogen; and R is: hydrogen, (C₁-C₆)-alkyl,(C₁-C₆)-alkylcarbonyl, phosphoryl, or polyalkoxy; or R and R¹ with theatoms to which they are attached form an optionally substituted ring;and single stereoisomers, mixtures of stereoisomers, and thepharmaceutically acceptable salts thereof.
 37. The method of claim 36,wherein R¹ is halogen, R⁴ is (optionallysubstituted(C₁-C₆)-alkyl)carbonyl and R⁵ is hydrogen.
 38. The method ofclaim 36, wherein the condition is selected from Alzheimer's disease,Parkinson's disease, Friedreich's ataxia, cerebellar ataxias, Leber'shereditary optic neuropathy, epilepsy, and myodegenerative disorders.39. The method of claim 38, wherein the condition is epilepsy.
 40. Themethod of claim 38, wherein the condition is Parkinson's disease. 41.The method of claim 38, wherein the condition is Friedreich's ataxia.42. A method of protecting cellular mitochondrial function against atoxic insult with compounds of Formula I:

wherein: R¹ is: hydrogen, optionally substituted (C₁-C₆)-alkyl,optionally substituted (C₂-C₁₀)-alkenyl, optionally substituted aryl,(optionally substituted (C₁-C₆)-alkoxy)carbonyl, or halogen; R² and R³are independently selected from optionally substituted (C₁-C₆)-alkyl,optionally substituted (C₂-C₁₀)-alkenyl, or optionally substituted(C₃-C₈)-cycloalkyl; R⁴ is: hydrogen, optionally substituted aryl,(optionally substituted(C₁-C₆)-alkyl)carbonyl, (optionally substitutedaryl)carbonyl, (optionally substituted heterocyclyl)carbonyl,(optionally substituted heterocyclylalkyl)carbonyl, (optionallysubstituted (C₁-C₆)-alkoxy)carbonyl, (optionally substituted(C₂-C₁₀)-alkenyloxy)carbonyl, (optionally substituted amino)carbonyl,carboxy, formyl, or hydroxy(optionally substituted)(C₁-C₆)-alkyl; R⁵ is:hydrogen, (C₁-C₆)-alkyl, (C₂-C₁₀)-alkenyl, (optionally substitutedalkoxy)carbonyl, carboxy, (optionally substituted amino)carbonyl, oroptionally substituted aryl; provided that one of R⁴ or R⁵ is hydrogen,and that when R⁴ is hydrogen R⁵ is not hydrogen, and when R⁵ is hydrogenR⁴ is not hydrogen; and R is: hydrogen, (C₁-C₆)-alkyl,(C₁-C₆)-alkylcarbonyl, phosphoryl, or polyalkoxy; or R and R¹ with theatoms to which they are attached form an optionally substituted ring;and single stereoisomers, mixtures of stereoisomers, and thepharmaceutically acceptable salts thereof.
 43. The method of claim 42,wherein said cellular mitochondrial function is in a neuronal cell. 44.The method of claim 43, wherein said neuronal cell is dopaminergic cell.45. The method of claim 44, wherein said dopaminergic cells are in theneurons of the substantia nigra-pars compacta.